Inhibition of tumor metastasis

ABSTRACT

The invention provides Nrp2 antagonists, such as anti-Nrp2 antibodies, and their use in the prevention and treatment of tumor metastasis.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.12/598,537, filed Mar. 19, 2010, claiming the benefit under 35 USC §371of PCT/US2007/069179, filed May 17, 2007. The disclosures of theforegoing applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention concerns neuropilin-2 (Nrp2) antagonists,especially anti-Nrp2 antibodies, and their use in the prevention andtreatment of tumor metastasis.

BACKGROUND OF THE INVENTION

It is now well established that angiogenesis is implicated in thepathogenesis of a variety of disorders. These include solid tumors andmetastasis, atherosclerosis, retrolental fibroplasia, hemangiomas,chronic inflammation, intraocular neovascular diseases such asproliferative retinopathies, e.g., diabetic retinopathy, age-relatedmacular degeneration (AMD), neovascular glaucoma, immune rejection oftransplanted corneal tissue and other tissues, rheumatoid arthritis, andpsoriasis. Folkman et al., J. Biol. Chem., 267:10931-10934 (1992);Klagsbrun et al., Annu. Rev. Physiol. 53:217-239 (1991); and Garner A.,“Vascular diseases”, In: Pathobiology of Ocular Disease. A DynamicApproach, Garner A., Klintworth G K, eds., 2nd Edition (Marcel Dekker,NY, 1994), pp 1625-1710.

In the case of tumor growth, angiogenesis appears to be crucial for thetransition from hyperplasia to neoplasia, and for providing nourishmentfor the growth and metastasis of the tumor. Folkman et al., Nature339:58 (1989). The neovascularization allows the tumor cells to acquirea growth advantage and proliferative autonomy compared to the normalcells. A tumor usually begins as a single aberrant cell which canproliferate only to a size of a few cubic millimeters due to thedistance from available capillary beds, and it can stay ‘dormant’without further growth and dissemination for a long period of time. Sometumor cells then switch to the angiogenic phenotype to activateendothelial cells, which proliferate and mature into new capillary bloodvessels. These newly formed blood vessels not only allow for continuedgrowth of the primary tumor, but also for the dissemination andrecolonization of metastatic tumor cells. Accordingly, a correlation hasbeen observed between density of microvessels in tumor sections andpatient survival in breast cancer as well as in several other tumors.Weidner et al., N Engl. J. Med 324:1-6 (1991); Horak et al., Lancet340:1120-1124 (1992); Macchiarini et al., Lancet 340:145-146 (1992). Theprecise mechanisms that control the angiogenic switch is not wellunderstood, but it is believed that neovascularization of tumor massresults from the net balance of a multitude of angiogenesis stimulatorsand inhibitors (Folkman Nat Med 1(1):27-31 (1995)).

It is currently accepted that metastases are responsible for the vastmajority, estimated at 90%, of deaths from solid tumors (Gupta andMassague, Cell 127, 679-695 (2006)). The complex process of metastasisinvolves a series of distinct steps including detachment of tumor cellsfrom the primary tumor, intravasation of tumor cells into lymphatic orblood vessels, and extravasation and growth of tumor cells in secondarysites. Analysis of regional lymph nodes in many tumor types suggeststhat the lymphatic vasculature is an important route for thedissemination of human cancers. Furthermore, in almost all carcinomas,the presence of tumor cells in lymph nodes is the most important adverseprognostic factor. While it was previously thought that such metastasesexclusively involved passage of malignant cells along pre-existinglymphatic vessels near tumors, recent experimental studies andclinicopathological reports (reviewed in Achen et al., Br J Cancer 94(2006), 1355-1360 and Nathanson, Cancer 98, 413-423 (2003)) suggest thatlymphangiogenesis can be induced by solid tumors and can promote tumorspread. These and other recent studies suggest targeting lymphatics andlymphangiogenesis may be a useful therapeutic strategy to restrict thedevelopment of cancer metastasis, which would have a significant benefitfor many patients.

VEGFC, a member of the vascular endothelial cell factor (VEGF) family,is one of the best studied mediators of lymphatic development.Overexpression of VEGFC in tumor cells was shown to promotetumor-associated lymphangiogenesis, resulting in enhanced metastasis toregional lymph nodes (Karpanen et al., Faseb J 20, 1462-1472 (2001);Mandriota et al., EMBO J 20, 672-682 (2001); Skobe et al., Nat Med 7,192-198 (2001); Stacker et al., Nat Rev Cancer 2, 573-583 (2002);Stacker et al., Faseb J 16, 922-934 (2002)). VEGFC expression has alsobeen correlated with tumor-associated lymphangiogenesis and lymph nodemetastasis for a number of human cancers (reviewed in Achen et al.,2006, supra. In addition, blockade of VEGFC-mediated signaling has beenshown to suppress tumor lymphangiogenesis and lymph node metastases inmice (Chen et al., Cancer Res 65, 9004-9011 (2005); He et al., J. NatlCancer Inst 94, 8190825 (2002); Krishnan et al., Cancer Res 63, 713-722(2003); Lin et al., Cancer Res 65, 6901-6909 (2005)).

VEGFC is known to bind at least two cell surface receptor families, thetyrosine kinase VEGF receptors and the neuropilin (Nrp) receptors.

Of the three VEGF receptors, VEGFC can bind VEGFR2 and VEGFR3 leading toreceptor dimerization (Shinkai et al., J Biol Chem 273, 31283-31288(1998)), kinase activation and autophosphorylation (Heldin, Cell 80,213-223 (1995); Waltenberger et al., J. Biol Chem 269, 26988-26995(1994)). The phosphorylated receptor induces the activation of multiplesubstrates leading to angiogenesis and lymphangiogenesis (Ferrara etal., Nat Med 9, 669-676 (2003)).

The neuropilin (Nrp) family is comprised of two homologous proteins,neuropilin-1 (Nrp1) and neuropilin-2 (Nrp2). In addition to the VEGFreceptors, VEGFC also binds to Nrp2, which was initially identified asclass 3 semaphorin receptor and mediator of axon guidance (Favier etal., Blood 108, 1243-1250 (2006); Soker et al., J Cell Biochem 85,357-368 (2002)). Multiple lines of evidence implicate Nrp2 in thedevelopment of the vascular and lymphatic systems. Homozygous Nrp2mutants show a severe reduction of small lymphatic vessels andcapillaries prenatally (Yuan et al., Development 129, 4797-4806 (2002)).Furthermore, the dramatic and embryonic lethal vascular defect seen inhomozygous Nrp1 mutant mice is enhanced by loss of Nrp2 function leadingto earlier lethality (Takashima et al., Proc Natl Acad Sci USA 99,3657-3662 (2002)). However, the role of Nrp2 in modulating adultvascular and lymphatic biology, and more specifically metastasis isunknown.

Nrps have short intracellular domains that are not known to have anyenzymatic or signaling activity. It has been proposed that Nrps functionto enhance VEGFR signaling by enhancing ligand-VEGF receptor binding(Favier et al., 2006, supra; Soker et al., 2002, supra). Additionally,sema3F, the semaphorin ligand of Nrp2, has been shown to modulateendothelial cell behavior in vitro and in vivo (Bielenberg et al., JClin Invest 114, 1260-1271 (2004); Favier et al., Blood 1243-1250,(2006)). However, recent reports have suggested an alternate possibilitythat Nrps may function independently of VEGF receptors or semaphorinfunction to modulate endothelial cell (EC) migration (Murga et al.,Blood 105, 1992-1999 (2005); Pan et al., Cancer Cell 11, 53-67 (2007);Wang et al., J Biol Chem 278, 48848-48860 (2003)).

Anti-VEGF neutralizing antibodies suppress the growth of a variety ofhuman tumor cell lines in nude mice (Kim et al., Nature 362:841-844(1993); Warren et al., J. Clin. Invest. 95:1789-1797 (1995); Borgströmet al., Cancer Res. 56:4032-4039 (1996); Melnyk et al., Cancer Res.56:921-924 (1996)) and also inhibit intraocular angiogenesis in modelsof ischemic retinal disorders. Adamis et al., Arch. Ophthalmol.114:66-71 (1996). Therefore, anti-VEGF monoclonal antibodies or otherinhibitors of VEGF action are promising candidates for the treatment oftumors and various intraocular neovascular disorders. Such antibodiesare described, for example, in EP 817,648 published Jan. 14, 1998; andin WO98/45331 and WO98/45332, both published Oct. 15, 1998. One of theanti-VEGF antibodies, bevacizumab, has been approved by the FDA for usein combination with a chemotherapy regimen to treat metastaticcolorectal cancer (CRC) and non-samll cell lung cancer (NSCLC). Andbevacizumab is being investigated in many ongoing clinical trials fortreating various cancer indications.

Other anti-VEGF antibodies and anti-Nrp1 antibodies are also known, anddescribed, for example, in Liang et al., J Mol Biol 366, 815-829 (2007);Pan et al., Cancer Cell 11, 53-67 (2007; and Liang et al., J Biol Chem281, 951-961 (2006)).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on experimentalresults obtained with a high-affinity function-blocking antibody toNrp2. Results obtained with this antibody indicate that Nrp2 plays arole in modulating lymphatic endothelial cell (LEC) migration, and thatits function extends beyond its previously assigned role as an enhancerof VEGF receptor activation. In addition, the results demonstrate thatblocking Nrp2 leads to an inhibition of lymphangiogenesis and a dramaticreduction in lymph node and distal organ metastasis.

In one aspect, the invention concerns a method for inhibiting lymphaticendothelial cell migration, comprising administering to a mammaliansubject in need an effective amount of a neuropilin-2 (Nrp2) antagonist.

In another aspect, the invention concerns a method for inhibitingtumoral lymphangiogenesis, comprising administering to a tumor-bearingmammalian subject an effective amount of a neuropilin-2 (Nrp2)antagonist.

In yet another aspect, the invention concerns a method for inhibitingtumor metastasis, comprising administering to a tumor-bearing mammaliansubject an effective amount of a neuropilin-2 (Nrp2) antagonist.

In all embodiments, the mammalian subject preferably is a human patient,such as a human cancer patient, who may have been diagnosed or may be atrisk of developing metastasis.

In one embodiment, the cancer is selected from the group consisting ofcarcinoma, lymphoma, blastoma, sarcoma, and leukemia.

In another embodiment, the cancer is selected from the group consistingof squamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, B-cell lymphoma,chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);Hairy cell leukemia; chronic myeloblastic leukemia; post-transplantlymphoproliferative disorder (PTLD), abnormal vascular proliferationassociated with phakomatoses, edema associated with brain tumors, andMeigs' syndrome.

In yet another embodiment, B-cell lymphoma is selected from the groupconsisting of low grade/follicular non-Hodgkin's lymphoma (NHL); smalllymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediategrade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia.

Without limitation, the Nrp2 antagonist can be an anti-Nrp2 antibody,including anti-Nrp2B and anti-Nrp2A antibodies, such as, for example,antibodies YW68.4.2, YW68.4.2.36, YW126.20, and fragments and variants,such as affinity matured variants thereof.

In another aspect, the invention concerns an anti-Nrp2B antibodycomprising the heavy and/or light chain variable region sequence of anantibody selected from the group consisting of YW68.4.2, YW68.4.2.36,and a fragment or variant thereof.

In yet another aspect, the invention concerns an anti-Nrp2A antibodycomprising the heavy and/or light chain variable region sequences ofYW126.20, or a fragment or variant thereof.

The invention further concerns a composition comprising an antibody ofany one of claims 33-38, in admixture with a pharmaceutically acceptablecarrier.

In a further aspect, the invention concerns a pharmaceutical compositionfor the prevention or treatment of tumor metastasis comprising aneffective amount of an Nrp2 antagonist in admixture with apharmaceutically acceptable carrier. In other aspects, the inventionconcerns Nrp2 antagonists for use in the prevention or treatment oftumor metastasis, and the use of Nrp2 antagonists, such as anti-Nrp2antibodies in the prevention or treatment of tumor metastasis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Characterization of Anti-Nrp2^(B) mAb. (A) Schematicrepresentation of Sema and VEGF binding regions on Nrp2 relative toAnti-Nrp2^(B) epitope regions. (B) ELISA assay demonstrating binding ofAnti-Nrp2^(B) to hNrp2 ECD (filled squares) and B1-B2 domains of hNrp2(filled circles), but not hNrp1 ECD (open squares) or the A1-A2 domainsof hNrp2 (open circles). (C) Blocking of VEGFC binding to Nrp2 byAnti-Nrp2^(B). Increasing amounts of mAb was pre-incubated with platescoated with human Nrp2 ECD (5 μg/ml) for 1-2 hrs, followed by addingpre-titrated biotinylated human VEGFC (1 nM) for 15 min. The percentageof bound VEGFC was detected by streptavidin-HRP conjugates. (D) Blockingof VEGF₁₆₅ binding to Nrp2 by Anti-Nrp2^(B). (E) Blocking of Sema3Fbinding to LECs. LEC were incubated with conditioned media containingSema3F fused to alkaline phosphatase (AP) (REF), in the presence orabsence of Anti-Nrp2^(B). AP activity derived form bound Sema3F-AP wasdetected colorimetrically with same development times. No binding wasobserved with AP (left panel). Anti-Nrp2^(B) did not block Sema3Fbinding to LECs (middle panels). Nrp2 ECD was used as a positive controlfor blocking binding (right panel). Scale bar

FIG. 2. Anti-Nrp2^(B) reduces VEGFC-induced function in vitro and invivo. (A) Representative images of stained LECs migrating in response to200 ng/ml of VEGFC for 18 hours in the presence or absence ofAnti-Nrp2^(B) (50 μg/ml) or VEGFR3 ECD (50 μg/ml). (B) Quantification ofLEC migration in response to 200 ng/ml VEGFC (n=6 for each condition).(C) Quantification of LEC migration in response to 10 ng/ml VEGF₁₆₅ inthe presence or absence of Anti-Nrp2^(B) (50 μg/ml) or VEGFR3ECD (50μg/ml). N=6 for each condition. (D) Quantification of the pixel countsfrom a corneal micropocket assay described in (E). *p<0.05 (E)Representative images of LYVE-1 stained cornea, illustrating the effectsof intracorneal placement of 150 ng pellet of VEGFC (P) and systemictreatment with Anti-Nrp2^(B) (10 mg/kg twice weekly) or VEGFR3ECD (25mg/kg twice weekly). LYVE-1 staining has been pseudocolored red tofacilitate visualization. *p<0.05; Error bars represent standard errorof the mean.

FIG. 3. Nrp2^(B) treatment results in a reduction in VEGF receptoractivation and inhibits Nrp2/VEGF receptor complex formation. (A) FACSanalysis of Nrp2, VEGFR2 ad VEGFR3 levels on the surface of LEC aftertreatment with control antibody (10 μg/ml; green line) or Anti-Nrp2^(B)(10 μg/ml) for 5 minutes (blue line), or 20 hours (red line). (B)Quantification of LEC migration in response to 20 ng/ml HGF in thepresence or absence of Anti-Nrp2^(B) (50 μg/ml) or VEGFR3ECD (50 μg/ml).N=6 for each condition. (C) VEGFR2 phosphorylation level in LECsdetected by ELISA assay using antibodies that recognized total ortyrosine-phosphorylated VEGFR2. VEGFC (concentration as noted) was addedfor 10 min in the presence or absence of Anti-Nrp2^(B) (10 μg/ml) orVEGFR3ECD (10 μg/ml) to induce the phosphorylation of VEGFR2, n=3 foreach condition. VEGFR2 phosphorylation level in anti-Nrp2^(B) (10 μg/ml)treated cells was significantly different from the VEGFC stimulation at200 ng/ml and consistently lay between the phosphorylation level inducedby 175 ng/nl and 150 ng/ml of VEGFC. (D) Quantification of LEC migrationin response to VEGFC (concentration as noted) in the presence or absenceof Anti-Nrp2^(B) (10 μg/ml) or VEGFR3ECD (10 μg/ml). Significantreductions in migration were noted at 50 ng/ml of VEGFC or when blockedwith VEGFR3 ECD. *p<0.05; Error bars represent the standard error of themean. Each experiment was repeated a minimum of three times. (E) CO-IP

FIG. 4. Nrp2 is expressed in the lymphatics of tumor bearing mice. (A-D)LYVE-1 staining (left column—red) labeling lymphatics, Nrp2 staining(middle column—green) and the overlay in the (A) intestine and (B) lymphnode of normal adult mouse. Nrp2 signal does not co-localize with LYVE-1labeled lymphatics in either organ. Rare Nrp2 staining inflammatorycells are present within the fibrostromal core of the intestinal villiand within the lymph node germinal centers (C) In lymph nodes from tumorbearing animals, Nrp2 signal does co-localize with LYVE-1 positivelymphatic vessels lining the LN sinuses. Additional Nrp2 staininginflammatory cells are also present. (D) Strong Nrp2 staining is alsoseen in lymphatic vessels within 66c14 tumors. (E) Weak membranousstaining can be also seen on tumor cells. Seconday only stained controlsdid not show any signal. Boxed areas are shown at high magnificationwithin insets. Scale bar *** for A-C and ** for D.

FIG. 5. Anti-Nrp2^(B) treatment results in a reduction of lungmetastasis in the 66c14 tumor model. (A) Mean tumor volume graph of66c14 tumor model study analyzed below. Animals were dosed twice weeklyi.p. with 10 mg/kg Anti-Nrp2^(B) or control antibody once tumors reachedan average size of 100 mm³ and were dosed throughout the study. (B)Quantification by visual inspection of the number of metastatic nodulesper lung in control and Anti-Nrp2^(B) treated animals. (C)Representative images of lungs from control (left) and Anti-Nrp2^(B)(right) treated animals. Lungs were inflated prior to fixation by rightcardiac ventricular perfusion. Nodules are highlighted in white tofacilitate visualization. (D, E) 3-dimendional renderings ofrepresentative micro-CT scanned lungs demonstrating metastatic nodules(red) in control (D) and Anti-Nrp2^(B) (E) treated animals. Thepositions of the longitudinal section (top inset) and the cross section(bottom insert) are indicated by the black and red dotted linesrespectively. This analysis confirms that most nodules are on thesurface of lungs. (F) Quantification of the number of metastatic nodulesper lung by Micro-CT analysis of the lungs. (G) FACS analysis of Nrp2levels on the surface of in vitro cultured 66c14 tumor cells. (H) H&Estaining of a lung nodule demonstrating metastatic tumor cells. Errorbars represent standard error of the mean. Scale bar *** for C and **for H.

FIG. 6. Anti-Nrp2^(B) treatment results in a reduction of lungmetastasis in the C6 tumor model. (A) Mean tumor volume graph of C6tumor model study analyzed below. Animals were dosed twice weekly i.p.with Anti-Nrp2^(B) (10 mg/kg), VEGFR3ECD (25 mg/kg) or control antibody(10 mg/kg) once tumors reached an average size of 100 mm³ and were dosedthroughout the study. (B) Quantification by visual inspection of thenumber of metastatic nodules per lung in control, VEGFR3ECD andAnti-Nrp2^(B) treated animals. (C) Representative images of lungs fromcontrol (left), VEGFR3ECD (middle) and Anti-Nrp2^(B) (right) treatedanimals. Lungs were inflated prior to fixation by right cardiacventricular perfusion. Nodules are highlighted in white to facilitatevisualization. (D) 3-dimendional renderings of representative micro-CTscanned lungs demonstrating metastatic nodules (red) in control (left)and Anti-Nrp2^(B) (right) treated animals. The positions of thelongitudinal section (top inset) and the cross section (bottom insert)are indicated by the black and red dotted lines respectively. Thisanalysis confirms that most nodules are on the surface of lungs. (E)FACS analysis of Nrp2 levels on the surface of in vitro cultured 66c14tumor cells. (F) H&E staining of a lung nodule demonstrating metastatictumor cells. Error bars represent standard error of the mean. Scale bar*** for C and ** for F.

FIG. 7. Anti-Nrp2^(B) treatment results in a reduction of tumorlymphatic vessels. (A, B) Quantification of vascular vessel density (A)as detected by PECAM-1 IHC and lymphatic vessel density (B) as detectedby LYVE-1 IHC in 66c14 tumors treated with control antibody orAnti-Nrp2^(B). Vessel density was determined from 6 representativeimages from each of 6 tumors per group, evaluated for mean pixel numberby ImageJ. (C) Representative mages of PECAM-1 stained vessels (top row)and LYVE-1 stained lymphatic vessels (middle and bottom rows) in C6tumors treated with control antibody (left column), VEGFR3ECD (middlecolumn) or Anti-Nrp2^(B) (right column). The boxed areas outlined in themiddle row are displayed in the bottom row at higher magnification.Quantification of vascular (top graph) and lymphatic (bottom graph)vessel density is to the right of these images. (D) LYVE-1 stainedtumors from Anti-Nrp2^(B) treated animals (bottom panels) harvested atday 4 (Harvest 1) and day 11 (Harvest 2) demonstrate disruption oflymphatic vessels in comparison to control treated animals (top panels).The harvest dates relative to growth curves are shown to the left. Errorbars represent standard error of the mean. Scale bar ***.

FIG. 8. Anti-Nrp2^(B) treatment results in a reduction of functionaltumor lymphatic vessels and leads to a delay in metastasis to theprimary lymph node. (A,B) Polystyrene fluorescent micro-beads (green)are seen exclusively in lymphatic vessels labeled by LYVE-1 IHC (red)after intradermal lymphangiography. The boxed area in A is shown athigher magnification in B. (C-D) Anti-Nrp2^(B) treatment results in areduction of evans blue within C6 (C) (P=0.035) and 66c14 (D) (P=0.005)tumors, indicating a reduction in functional lymphatics with in thesetreated tumors. (E) Percent of animals with SLNs containing β-galexpressing C6 tumor cells at various time-points after tumorimplantation in the ears of control (black) and Anti-Nrp2^(B) treated(red) mice. Anti-Nrp2^(B) treatment results in a delay of arrival ofcells at the SLN (p=0.006). N=7 animals per treatment condition pertime-point.

FIG. 9. Expression of Nrp2 in different human malignancies. (A-F)Affymetrix HG-U133A and B GeneChip® microarray data for Nrp2 expressionin normal colon and colorectal adenocarcinoma (A), normal head and necktissues and head and neck squamous cell carcinoma (B), normal pancreasand pancreatic adenocarcinoma (C), normal skin and malignant melanoma(D), normal thyroid and papillary throid carcinoma (E), and normalbreast and Her2-infiltrating ductal adenocarcinoma (F). Each datapointrepresents one patient.

FIG. 10. Amino acid sequences of anti-Nrp2^(B) antibody YW68.4.2 (A) andYW68.4.2.36 (B) SEQ ID NOs: 1 and 2, respectively.

FIG. 11. Amino acid sequence of anti-Nrp2^(A) antibody YW126.20 Fabfragment (SEQ ID NO: 3).

FIG. 12. Alignment of anti-Nrp2^(A) antibody YW126.20 light chainvariable domain sequence (SEQ ID NO: 5) with human κ1(1 sequence (SEQ IDNO: 4).

FIG. 13. Alignment of anti-Nrp2^(A) antibody YW126.20 heavy chainvariable domain sequence (SEQ ID NO: 7) with human III (hum III)sequence (SEQ ID NO: 6).

FIG. 14. FACS analysis of VEGF axis receptors' levels on the surface ofin vitro cultured LECs. FACS analysis of Nrp1, Nrp2, VEGFR1, VEGFR2 adVEGFR3 on cultured LECs.

FIG. 15. Anti-Nrp2^(B) does not block VEGF₁₆₅-induced migration. (A-B)Quantification of HUVEC (A) and LEC (B) migrating in response to 200ng/ml of VEGF for 18 hours in the presence or absence of Anti-Nrp1^(B),Anti-Nrp2^(B) (50 μg/ml) or both antibodies (50 μg/ml). *p<0.05; Errorbars represent standard error of the mean.

FIG. 16. Effects of Anti-Nrp2^(B) on VEGFC mediated proliferation andvascular permeability and associated intracellular signaling. (A)Quantification of LEC proliferation induced by 200 ng/ml VEGFC in thepresence or absence of Anti-Nrp2^(B) (50 μg/ml) or VEGFR3ECD (50 μg/ml)as determined by BrdU incorporation (n=6 per condition). (B) Mouse skinvascular permeability assay. Images were taken from the skin of the sameanimal Blue stain represents Evan's blue leakage from the vasculature inresponse to intradermal delivery of VEGFC after systemic treatment withAnti-Nrp2^(B) (10 mg/kg) or VEGFR3ECD (25 mg/kg). (C) Quantification ofthe Evan's blue dye extracted from skin samples in the permeabilityassay. Values shown are the average of 6 independent experiments.*p<0.05; Error bars represent standard error of the mean.

FIG. 17. Anti-Nrp2^(B) does not block Sema3F induced growth conecollapse. (A) Images of E 17.5 Hippocampal growth cones stained withrhodamine conjugated phalloidin. Control growth cones show large actinrich structures at the tip of each axon, which are reduced with Sema3Ftreatment. Anti-Nrp2^(B) (50 μg/ml) does not block this collapse. Incontrast, Nrp2 ECD (10 ug/ml) does block this collapse. (B) Qualitycontrol for anti-Nrp2 immunohistochemistry. Image shows a phage-derivedclone that recognized Nrp2 that works in IHC on fresh frozen sections.Nrp2 protein expression is similar to Nrp2 expression as seen using insitu hybridization (Chen et al., Neuron 19, 547-559 (1997)). Thisantibody was subsequently used for IHC on fresh frozen tumor sections.(Scale bar main images=**μm and for inset images=**μm.

FIG. 18. (A) Micro-CT images of lungs (B) Comparison of lung tumorvolume estimates.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “Neuropilin”, “NRP” or “Nrp” are used interchangeably andrefer collectively to neuropilin-1 (NRP1, Nrp1), neuropilin-2 (NRP2,Nrp2) and their isoforms and variants, as described in Rossignol et al.(2000) Genomics 70:211-222. Neuropilins are 120 to 130 kDa non-tyrosinekinase receptors. There are multiple NRP-1 and NRP-2 splice variants andsoluble isoforms. The basic structure of neuropilins comprises fivedomains: three extracellular domains (a1a2, b1b2 and c), a transmembranedomain, and a cytoplasmic domain. The a1a2 domain is homologous tocomplement components C1r and C1s (CUB), which generally contains fourcysteine residues that form two disculfid bridges. The b1b2 domain ishomologous to coagulation factors V and VIII. The central portion of thec domain is designated as MAM due to its homology to meprin, A5 andreceptor tyrosine phosphotase μ proteins. The a1a2 and b1b2 domains areresponsible for ligand binding, whereas the c domain is critical forhomodimerization or heterodimerization. Gu et al. (2002) J. Biol. Chem.277:18069-76; He and Tessier-Lavigne (1997) Cell 90:739-51.

“Neuropilin mediated biological activity” refers in general tophysiological or pathological events in which neuropilin-1 and/orneuropilin-2 plays a substantial role. Non-limiting examples of suchactivities are axon guidance during embryonic nervous system developmentor neuron-regeneration, angiogenesis (including vascular modeling),tumorgenesis and tumor metastasis.

“Neuropilin-2 mediated biological activity” or “Nrp2 mediated biologicalactivity,” as used herein, refers in general to physiological orpathological events in which Nrp2 plays a substantial role, such as, forexample, enhancing VEGF receptor activation, and, in particular, theability to modulate lymphatic endothelial cell (EC) migration, role inadult lymphangiogenesis, especially tumoral lymphangiogenesis and tumormetastasis.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al. (1975)Nature 256:495, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al. (1991) Nature 352:624-628 and Marks et al. (1991) J.Mol. Biol. 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta(1992) Curr. Op. Struct. Biol. 2:593-596.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen (i.e.has a binding affinity (K_(d)) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ M and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but preferably is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Ina preferred embodiment, the antibody mutant will have an amino acidsequence having at least 75% amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the species-dependent antibody, more preferably at least 80%,more preferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e same residue) or similar(i.e. amino acid residue from the same group based on common side-chainproperties, see below) with the species-dependent antibody residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into theantibody sequence outside of the variable domain shall be construed asaffecting sequence identity or similarity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; ie., CDR1, CDR2,and CDR3), and Framework Regions (FRs). V_(H) refers to the variabledomain of the heavy chain. V_(L) refers to the variable domain of thelight chain. According to the methods used in this invention, the aminoacid positions assigned to CDRs and FRs may be defined according toKabat (Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991)). Amino acidnumbering of antibodies or antigen binding fragments is also accordingto that of Kabat.

As used herein, the term “Complementarity Determining Regions (CDRs;ie., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. For example, the CDRH1 of the heavy chain of antibody 4D5 includesamino acids 26 to 35.

“Framework regions” (hereinafter FR) are those variable domain residuesother than the CDR residues. Each variable domain typically has four FRsidentified as FR1, FR2, FR3 and FR4. If the CDRs are defined accordingto Kabat, the light chain FR residues are positioned at about residues1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and theheavy chain FR residues are positioned about at residues 1-30 (HCFR1),36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chainresidues. If the CDRs comprise amino acid residues from hypervariableloops, the light chain FR residues are positioned about at residues 1-25(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the lightchain and the heavy chain FR residues are positioned about at residues1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in theheavy chain residues. In some instances, when the CDR comprises aminoacids from both a CDR as defined by Kabat and those of a hypervariableloop, the FR residues will be adjusted accordingly. For example, whenCDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

As used herein, “codon set” refers to a set of different nucleotidetriplet sequences used to encode desired variant amino acids. A set ofoligonucleotides can be synthesized, for example, by solid phasesynthesis, including sequences that represent all possible combinationsof nucleotide triplets provided by the codon set and that will encodethe desired group of amino acids. A standard form of codon designationis that of the IUB code, which is known in the art and described herein.A codon set typically is represented by 3 capital letters in italics,eg. NNK, NNS, XYZ, DVK and the like. A “non-random codon set”, as usedherein, thus refers to a codon set that encodes select amino acids thatfulfill partially, preferably completely, the criteria for amino acidselection as described herein. Synthesis of oligonucleotides withselected nucleotide “degeneracy” at certain positions is well known inthat art, for example the TRIM approach (Knappek et al. (1999) J. Mol.Biol. 296:57-86); Garrard & Henner (1993) Gene 128:103). Such sets ofoligonucleotides having certain codon sets can be synthesized usingcommercial nucleic acid synthesizers (available from, for example,Applied Biosystems, Foster City, Calif.), or can be obtainedcommercially (for example, from Life Technologies, Rockville, Md.).Therefore, a set of oligonucleotides synthesized having a particularcodon set will typically include a plurality of oligonucleotides withdifferent sequences, the differences established by the codon set withinthe overall sequence. Oligonucleotides, as used according to theinvention, have sequences that allow for hybridization to a variabledomain nucleic acid template and also can, but does not necessarily,include restriction enzyme sites useful for, for example, cloningpurposes.

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimerCollectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (CH1) of theheavy chain. F(ab′)₂ antibody fragments comprise a pair of Fab fragmentswhich are generally covalently linked near their carboxy termini byhinge cysteines between them. Other chemical couplings of antibodyfragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman (1992) Curr. Opin.Struct. Biol. 3:355-362, and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells (1991) Methods: A companion to Methodsin Enzymology 3:205-0216.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, fl, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

As used herein, “solvent accessible position” refers to a position of anamino acid residue in the variable regions of the heavy and light chainsof a source antibody or antigen binding fragment that is determined,based on structure, ensemble of structures and/or modeled structure ofthe antibody or antigen binding fragment, as potentially available forsolvent access and/or contact with a molecule, such as anantibody-specific antigen. These positions are typically found in theCDRs and on the exterior of the protein. The solvent accessiblepositions of an antibody or antigen binding fragment, as defined herein,can be determined using any of a number of algorithms known in the art.Preferably, solvent accessible positions are determined usingcoordinates from a 3-dimensional model of an antibody, preferably usinga computer program such as the InsightII program (Accelrys, San Diego,Calif.). Solvent accessible positions can also be determined usingalgorithms known in the art (e.g., Lee and Richards (1971) J. Mol. Biol.55, 379 and Connolly (1983) J. Appl. Cryst. 16, 548). Determination ofsolvent accessible positions can be performed using software suitablefor protein modeling and 3-dimensional structural information obtainedfrom an antibody. Software that can be utilized for these purposesincludes SYBYL Biopolymer Module software (Tripos Associates). Generallyand preferably, where an algorithm (program) requires a user input sizeparameter, the “size” of a probe which is used in the calculation is setat about 1.4 Angstrom or smaller in radius. In addition, determinationof solvent accessible regions and area methods using software forpersonal computers has been described by Pacios (1994) Comput. Chem.18(4): 377-386.

An “angiogenic factor or agent” is a growth factor which stimulates thedevelopment of blood vessels, e.g., promote angiogenesis, endothelialcell growth, stabiliy of blood vessels, and/or vasculogenesis, etc. Forexample, angiogenic factors, include, but are not limited to, e.g., VEGFand members of the VEGF family, P1GF, PDGF family, fibroblast growthfactor family (FGFs), TIE ligands (Angiopoietins), ephrins, Del-1,fibroblast growth factors: acidic (aFGF) and basic (bFGF), Follistatin,Granulocyte colony-stimulating factor (G-CSF), Hepatocyte growth factor(HGF)/scatter factor (SF), Interleukin-8 (IL-8), Leptin, Midkine,neuropilins, Placental growth factor, Platelet-derived endothelial cellgrowth factor (PD-ECGF), Platelet-derived growth factor, especiallyPDGF-BB or PDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin,Transforming growth factor-alpha (TGF-alpha), Transforming growthfactor-beta (TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), etc. Itwould also include factors that accelerate wound healing, such as growthhormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growthfactor (EGF), CTGF and members of its family, and TGF-alpha andTGF-beta. See, e.g., Klagsbrun and D'Amore (1991) Annu. Rev. Physiol.53:217-39; Streit and Detmar (2003) Oncogene 22:3172-3179; Ferrara &Alitalo (1999) Nature Medicine 5(12):1359-1364; Tonini et al. (2003)Oncogene 22:6549-6556 (e.g., Table 1 listing known angiogenic factors);and, Sato (2003) Int. J. Clin. Oncol. 8:200-206.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, an polynucleotide, an polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as defined above,e.g., antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptoror Flt-1 receptor), anti-PDGFR inhibitors such as Gleevec™ (ImatinibMesylate). Anti-angiogensis agents also include native angiogenesisinhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun andD'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003)Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy inmalignant melanoma); Ferrara & Alitalo (1999) Nature Medicine5(12):1359-1364; Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table2 listing known antiangiogenic factors); and, Sato (2003) Int. J. Clin.Oncol. 8:200-206 (e.g., Table 1 listing anti-angiogenic agents used inclinical trials).

The term “VEGF” or “VEGF-A” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. (1989) Science 246:1306, and Houck et al.(1991) Mol. Endocrin, 5:1806, together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109)” or “VEGF₁₆₅.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. Preferably, the anti-VEGF antibodyof the invention can be used as a therapeutic agent in targeting andinterfering with diseases or conditions wherein the VEGF activity isinvolved. An anti-VEGF antibody will usually not bind to other VEGFhomologues such as VEGF-B or VEGF-C, nor other growth factors such asP1GF, PDGF or bFGF. A preferred anti-VEGF antibody is a monoclonalantibody that binds to the same epitope as the monoclonal anti-VEGFantibody A4.6.1 produced by hybridoma ATCC HB 10709. More preferably theanti-VEGF antibody is a recombinant humanized anti-VEGF monoclonalantibody generated according to Presta et al. (1997) Cancer Res.57:4593-4599, including but not limited to the antibody known asbevacizumab (BV; Avastin™)

The anti-VEGF antibody “Bevacizumab (BV)”, also known as “rhuMAb VEGF”or “Avastin®, is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated.

The terms “VEGFC” and “VEGF-C” are used interchangeably, and refer to a419-amino acid human polypeptide (SwissProt: VEGFC_HUMAN P49767), andnon-human mammalian orthologs thereof, first described by Joukov et al.,EMBO J. 15, 290-98 (1996), and EMBO J. 15, 1751 (1996).

The term “Nrp2 antagonist” is used herein to refer to a molecule capableof neutralizing, blocking, inhibiting, abrogating, reducing orinterfering with the ability of Nrp2 to modulate lymphatic endothelialcell (EC) migration, or adult lymphangiogenesis, especially tumorallymphangiogenesis and tumor metastasis.

A “VEGF antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with VEGFactivities including, but not limited to, its binding to one or moreVEGF receptors. VEGF antagonists include, without limitation, anti-VEGFantibodies and antigen-binding fragments thereof, receptor molecules andderivatives which bind specifically to VEGF thereby sequestering itsbinding to one or more receptors, anti-VEGF receptor antibodies and VEGFreceptor antagonists such as small molecule inhibitors of the VEGFRtyrosine kinases. The term “VEGF antagonist,” as used herein,specifically includes molecules, including antibodies, antibodyfragments, other binding polypeptides, peptides, and non-peptide smallmolecules, that bind to neuropilin-1 and/or neuropilin-2 (Nip-1 and/orNrp-2) and are capable of neutralizing, blocking, inhibiting,abrogating, reducing or interfering with VEGF activities including, butnot limited to, anti-Nrp1 and anti-Nrp2 antibodies and antibodiescross-reacting with Nrp1 and Nrp2, provided they are capable ofneutralizing, blocking, inhibiting, abrogating, reducing or interferingwith VEGF activities. Thus, the term “VEGF activities” specificallyincludes neuropilin mediated biological activities (as hereinabovedefined) of VEGF.

A “semaphorin antagonists” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering withsemaphorin activities including, but not limited to, its binding to oneor more semaphorin receptors. Semaphorin antagonists include, withoutlimitation, anti-semaphorin antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives which bind specifically tosemaphorin thereby sequestering its binding to one or more receptors,anti-semaphorin receptor antibodies and semaphorin receptor antagonistssuch as small molecule inhibitors of semaphorins. The term “semaphorinantagonist,” as used herein, specifically includes molecules, includingantibodies, antibody fragments, other binding polypeptides, peptides,and non-peptide small molecules, that bind to neuropilin-1 and/orneuropilin-2 (Nrp-1 and/or Nip-2) and are capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering withsemaphorin activities including, but not limited to, anti-Nrp1 andanti-Nrp2 antibodies and antibodies cross-reacting with Nrp1 and Nrp2,provided they are capable of neutralizing, blocking, inhibiting,abrogating, reducing or interfering with semaphorin activities. Thus,the term “semaphorin activities” specifically includes neuropilinmediated biological activities (as hereinabove defined) of class 3semaphorins. Such biological activities include, for example, neuritegrowth inhibitory effect during embryonic nervous system development andneuron-regeneration.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatment. Forexample, mammals who suffer from or need prophylaxis against abnormalangiogenesis (excessive, inappropriate or uncontrolled angiogenesis) orvascular permeability. This includes chronic and acute disorders ordiseases including those pathological conditions which predispose themammal to the disorder in question. Non-limiting examples of disordersto be treated herein include malignant and benign tumors; non-leukemiasand lymphoid malignancies; and, in particular, tumor (cancer)metastasis.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

The term “anti-neoplastic composition” refers to a composition useful intreating cancer comprising at least one active therapeutic agent, e.g.,“anti-cancer agent.” Examples of therapeutic agents (anti-cancer agents)include, but are limited to, e.g., chemotherapeutic agents, growthinhibitory agents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g.,a tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib(Tarceva™), platelet derived growth factor inhibitors (e.g., Gleevec™(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons,cytokines, antagonists (e.g., neutralizing antibodies) that bind to oneor more of the following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, B1yS,APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2, and other bioactive andorganic chemical agents, etc. Combinations thereof are also included inthe invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g., I¹³¹,I¹²⁵, Y⁹⁰ and Re¹⁸⁶), chemotherapeutic agents, and toxins such asenzymatically active toxins of bacterial, fungal, plant or animalorigin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include is achemical compound useful in the treatment of cancer. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andCYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew (1994) Chem. Intl. Ed. Engl. 33:183-186);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)(including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,erlotinib (Tarceva™)) and VEGF-A that reduce cell proliferation andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON.toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No.4,675,187), and pharmaceutically acceptable salts, acids or derivativesof any of the above.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman (1986) “Prodrugs in Cancer Chemotherapy”Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfastand Stella et al. (1985). “Prodrugs: A Chemical Approach to TargetedDrug Delivery,” Directed Drug Delivery, Borchardt et al, (ed.), pp.247-267, Humana Press. The prodrugs of this invention include, but arenot limited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

MODES FOR CARRYING OUT THE INVENTION

In one aspect, the present invention is based on experimental datademonstrating that blocking Nrp2 function inhibits tumor metastasis.

A key event in the multi-step process of metastasis involves the egressof a tumor cell away from the primary tumor mass. For solid tumors, thelymphatic system often provides a route for the departing cells. VEGF isknown to be a key modulator of lymphangiogenesis and metastasis in manytumor models, and inhibition of the VEGF axis is considered a promisingstrategy for inhibiting the development of metastasis. Before thepresent invention, Nrp2, a co-receptor for VEGFC, has not been deemed atarget for inhibiting tumor metastasis, possibly due to the lack oflymphatic system defects in adult Nrp2 mutant mice.

The studies underlying the present invention, which are presented in theexamples below, support an important role of Nrp2 in tumorlymphangiogenesis and metastasis, only in part by modulating VEGFR3signaling. Additionally, the data set forth in the Examples demonstratethe presence of functional lymphatic vessels within tumors and show thattreating with Anti-Nrp2^(B) results in a reduction of these functionallymphatics.

Nrp2 Regulated Selective VEGFC Functions, in Part Through a MechanismIndependent of VEGF Receptor Activation

Induction of cellular migration and proliferation are two of the centralcellular functions of VEGFC described to date (Joukov et al., Embo J 16,3898-3911 (1997)). Thus, the present finding that blocking Nrp2 withAnti-Nrp2^(B) blocked LEC migration but not proliferation (FIG. 2, 3)was surprising. This selectivity has been recently reported with Nrp2siRNA knockdown experiments, but was attributed to experimentaltechnical limitations (Favier et al., Blood 108, 1243-1250 (2006)). Thedata presented herein show that Nrp2's functional selectivity was alsonoted in vivo, where Anti-Nrp2^(B) treatment resulted in a reduction ofVEGFC driven lymphangiogenesis but not vascular permeability (FIG. 2,3). These observations suggest that inhibition with Anti-Nrp2^(B) doesnot simply function by disrupting VEGFC signaling. However, it has beendetermined that blocking Nrp2 did result in a modest reduction in VEGFreceptor phosphorylation (FIG. 3) supporting a mechanism where one ofNrp2's roles is to enhance VEGF receptor function. This raised thepossibility that different VEGFC-induced physiological events mayrequire different levels of VEGF receptor activation. Thus, a decreasein receptor activation may be sufficient to affect migration, but notproliferation or vascular permeability.

To test this, the VEGFC dose response of VEGF receptor phosphorylationwas compared to the dose response of LEC migration (FIG. 3). Doses ofVEGFC that led to a receptor phosphorylation level equivalent to thatseen with Anti-Nrp2^(B) treatment did not reduce or inhibit migration.This indicated that the decrease in receptor activation alone did notaccount for the function blocking effects of Anti-Nrp2^(B).

Therefore, other mechanisms were investigated by which blocking Nrp2 mayselectively affect migration such as modulation of adhesion or motility.Anti-Nrp2^(B) treatment did not have any effect on LEC mediated adhesionor migration induced by VEGF₁₆₅ (FIG. 2), HGF (FIG. 3) or FGF-2,indicating that it did not generally affect migration by disruptingmotility. Additionally, it has been proposed that sema3F, another ligandof Nrp2, may modulate LEC or EC migration, acting as a chemorepellant(Bielenberg et al., J Clin Invest 114, 1260-1271 (2004); Favier et al.,Blood 108, 1243-1250 (2006)). However, the Anti-Nrp2^(B) antibody didnot inhibit or potentiate the binding of sema3F to Nrp2 (FIG. 1) or thefunctional effects of sema3F on responsive neurons (FIG. 17). Thus, itis unlikely that the reduction in VEGFC induced migration byAnti-Nrp2^(B) can be explained by modulation of sema3F function.

The effect of Anti-Nrp2^(B) on the formation of the Nrp2/VEGF receptorcomplex has also been evaluated. In contrast to Nrp1, Nrp2 forms acomplex with VEGFR2 and VEGFR3 in the absence of ligand (Favier et al.,2006, supra; Karpanen et al., Faseb J 20, 1462-1472 (2006)).Importantly, Anti-Nrp2^(B) strongly inhibits the formation of thesecomplexes. This observation, in addition to the fact that Nrp2 issignificant for more than just augmentation of VEGF receptor function,supports a model in which Nrp2 provides additional functionality tospecifically modulate migration, potentially conveying additionalmachinery to the VEGF receptor complex.

Nrp2 Plays an Important Role in Modulating Adult Lymphangiogenesis.

Analysis of Nrp2 KO mice demonstrates that Nrp2 is a modulator ofdevelopmental lymphangiogenesis, presumably via its role as a VEGFCco-receptor (Yuan et al., 2002, supra). However, these mutant mice formfunctional lymphatics after birth, indicating that either the defectrepresents a delay rather than inhibition of lymphatic growth or thatthere is functional compensation by another molecular mediator.Therefore, the role of Nrp2 in maintaining mature lymphatics andmodulating adult lymphangiogenesis has not been not known. Expressionanalysis (FIG. 4) does not support a role of Nrp2 in maintaininglymphatics. Interestingly, Nrp2 is strongly expressed in lymphatics thatare present in tumors and within LNs adjacent to tumors, suggesting thatNrp2 may play a role in activated or growing lymphatics. In vitroobservations demonstrate that Anti-Nrp2^(B) is an effective tool inevaluating the role of Nrp2 in these processes. Therefore, Anti-Nrp2^(B)was tested in vivo using the corneal micropocket assay (FIG. 2).Anti-Nrp2^(B) effectively blocked the VEGFC induced lymphangiogenesis,surprisingly equivalently to VEGFR3ECD. Interestingly, Anti-Nrp2^(B)demonstrated selective inhibitory function in vivo as well, failing toaffect VEGFC induced vascular permeability. This corresponds with the invitro observations that Nrp2 specifically modulates migration, a processimportant for lymphangiogenesis, but unlikely to play a role in vascularpermeability. Finally, these Anti-Nrp2^(B) treated normal adult animalsdid not demonstrate any changes to intestinal lymphatics, confirmingNrp2 does not play a role in maintenance of mature lymphatics.

Nrp2 Inhibition Leads to a Reduction in Functional Lymphatics within theTumor and a Reduction in Metastasis—Likely by Inhibiting Tumor Cellsfrom Leaving the Main Tumor Mass Via the Lymphatic Route.

Inhibition of the VEGFC axis, most often by the use of VEGFR3ECD, is oneof the more commonly utilized strategies for reducing metastasis (Chenet al., 2005. supra; He et al., 2002, supra; Krishnan et al., 2003,supra; Lin et al., 2005, supra). VEGFC can facilitate metastasispotentially by initiating lymphangiogenesis, thereby increasing thesurface area of tumor cells in contact with LECs, by modulating LECadhesive properties or cytokine expression or by increasing vascularpermeability (Alitalo and Carmeliet, Cancer Cell 1, 219-227 (2002)). AsAnti-Nrp2^(B) modulates selective VEGFC mediated functions includinginhibiting VEGFC induced adult lymphangiogenesis, next, the effects ofblocking Nrp2 on metastasis were investigated. In order to minimizeconfounding variables and to unambiguously evaluate the role ofAnti-Nrp2^(B) on metastasis, we picked models where blocking Nrp2 doesnot affect primary tumor growth and further harvested all animals at thesame time-point in the study (Withers and Lee, Semin Radiat Oncol 16,111-119 (2006)).

In both 66c14 as well as C6 tumor models, Anti-Nrp2^(B) treatmentresulted in a significant reduction of metastatic lung nodules by visualinspection (FIG. 5, 6). This was confirmed by the more sensitive andquantitative micro-CT technique (L1 et al., Technol Cancer Res Treat 5,147-155 (2006)). Comparison of Anti-Nrp2^(B) and VEGFR3ECD treatmentswas not possible in 66c14 tumors due to a reduction in primary tumorsize with VEGFR3 treatment. However, this analysis was conducted in C6tumors as their growth was not affected by VEGFR3ECD treatment. As withthe corneal micropocket assay, Anti-Nrp2^(B) treatment resulted in anequivalent block of metastasis when compared to VEGFR3ECD.

Histologic analysis of the primary tumor indicated that we were notprimarily affecting tumor cells. Thus, we evaluated the two potentialmetastatic routes that were available to tumor cells: blood vessels andlymphatics (FIG. 7). Treatment with Anti-Nrp2^(B) did not affect thestructure or density of blood vessels. Based on the in vitro and cornealmicropocket in vivo data, it was hypothesized that blocking Nrp2 shouldresult in a reduction of tumor lymphatics. Anti-Nrp2^(B) treatment diddramatically reduce the density of lymphatics, and again, to anequivalent degree as VEGFR3ECD treatment. However, these two treatmentsdid differ in the morphology of the resulting lymphatics. VEGFR3 ECDtreatment led to the formation of sparse lymphatic networks lined byunhealthy appearing lymphatic cells. Anti-Nrp2^(B) treatment, on theother hand, led to the development of short vessels and pockets ofisolated healthy appearing lymphatic cells. These differences furthersupport a model where Nrp2 does not simply act to augment VEGF receptoractivation, but also brings unique functionality to mediate VEGFCbiology. These results also demonstrate that for the experimentalparadigms tested, Anti-Nrp2^(B) acts to inhibit lymphangiogenesis (FIG.7). However, it cannot be ruled out that Anti-Nrp2^(B) also disruptsmore established lymphatic vessels within tumors.

We also sought to determine if intratumoral lymphatics were functionaland therefore competent to facilitate metastasis. Lymphangiography wasused to identify rare functional intratumoral lymphatics (FIG. 8). Thetechnique used was not adequately analytical to determine the proportionof intratumoral lymphatics that were functional. However, they arelikely to represent a small fraction of the total lymphatic population(Padera et al., Mol Imaging 1, 9-15 (2002)). As it was possible that wewere reducing total lymphatic density while sparing functional vessels(which may have different sensitivity to Anti-Nrp2^(B)), we evaluatedthe effects of blocking Nrp2 on the formation of functional lymphaticvessels. Anti-Nrp2^(B) reduced the formation of functional vessels,thereby more directly linking the effects on tumor lymphatics with theobserved reduction in metastasis.

Finally, to confirm the consequence of reducing functional lymphatics,the effects of Anti-Nrp2^(B) on metastasis to the SLN were evaluated.The SLN is the first tissue that tumor cells encounter after departingfrom the tumor via the lymphatics. Thus, it represents one of theearliest steps in distant organ metastasis (Stracke and Liotta, In Vivo6, 309-316 (1992)). As predicted, Anti-Nrp2^(B) treatment resulted in adelay of the development of SLN micrometastasis, consistent with theidea that fewer cells were effluxing from the primary tumor mass. Thisis consistent with evidence that VEGFC increases metastasis by inducinglymphatic hyperplasia and increased delivery of cancer cells to lymphnodes (Hoshida et al., Cancer Research 66, 8065-8075 (2006)). Thus, theweight of evidence points to a mechanism by which blocking Nrp2 leads toa reduction in functional tumor lymphatics, thereby preventing tumorcells from initiating the metastatic process by exiting from the primarytumor mass.

Nrp2 as a Metastasis Target.

Numerous clinico-pathologic studies have reported that expression ofVEGFC and VEGFR3 correlate with lymph node metastasis and distalmetastasis in a number of human cancers (extensively reviewed in Stackeret al., Nat Rev Cancer, 2002, supra; Stacker et al., Faseb J, 2002,supra, and He et al., 2004, supra). However, there is limitedinformation related to Nrp2 expression and its relation to metastasis.Indeed, links have only been made between Nrp2 expression andmalignancy, particularly in pancreatic cancer (Cohen et al., BiochemBiophys Res Commun 284, 395-403 (2001); Fukahi et al., Clin Cancer Res10, 581-590 (2004)) and lung cancer (Kawakami et al., Cancer 95,2196-2201 (2002); Lantuejoul et al., J Pathol 200, 336-347 (2003)). Itwas similarly found that Nrp2 was expressed at higher levels compared totheir respective control tissues, not only in pancreatic, but also incolonic adenocarcinoma, head and neck squamous cell carcinoma, melanoma,papillary thyroid carcinoma and infiltrating ductal adenocarcinoma ofthe breast (FIG. 9). More importantly, when these tumors were subdividedinto metastatic and non-metastatic groups, Nrp2 expression was noted tobe statistically higher in the metastatic group in most of these tumortypes. Interestingly, these tumor types all have confirmed intratumorallymphangiogenesis that furthermore has been correlated with lymph nodemetastasis (Achen et al., 2006, supra; Achen and Stacker, Int J Cancer119, 1755-1760 (2006)). This indicates that the discussed experimentalfindings are expected to extend to human patients with a variety oftumor types.

In conclusion, the data discussed herein and presented in the Examplesbelow show that Nrp2 plays a role in modulating VEGFC driven cellularmigration and provide evidence that Nrp2 may act through multiplemechanisms including enhancing VEGF receptor activation and mechanismsindependent of VEGF receptor signaling. Additionally, blocking Nrp2function using anti-Nrp2^(B) results in a dramatic reduction of VEGFCinduced lymphangiogenesis in adult mice. This treatment also results ina reduction of metastasis, likely via a reduction in the development offunctional lymphatics. These data, along with analysis of Nrp2expression in a number of human tumors, suggest that Nrp2 is a validtarget to modulate metastasis.

Production of Anti-Nrp2 Antibodies

The invention herein includes the production and use of anti-NRP2antibodies. Exemplary methods for generating antibodies are described inmore detail in the following sections.

Anti-NRP2 antibodies are selected using an NRP2 antigen derived from amammalian species. Preferably the antigen is human NRP2 (hNRP2).However, NRP2s from other species such as murine NRP2 (mNRP2) can alsobe used as the target antigen. The NRP2 antigens from various mammalianspecies may be isolated from natural sources. In other embodiments, theantigen is produced recombinantly or made using other synthetic methodsknown in the art.

The antibody selected will normally have a sufficiently strong bindingaffinity for the NRP2 antigen. For example, the antibody may bind hNRP2with a K_(d) value of no more than about 5 nM, preferably no more thanabout 2 nM, and more preferably no more than about 500 pM. Antibodyaffinities may be determined by a surface plasmon resonance based assay(such as the BIAcore assay as described in Examples); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample.

Also, the antibody may be subject to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody. Examples include the HUVEC inhibitionassay (as described in the Examples below); tumor cell growth inhibitionassays (as described in WO 89/06692, for example); antibody-dependentcellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC)assays (U.S. Pat. No. 5,500,362); and agonistic activity orhematopoiesis assays (see WO 95/27062).

To screen for antibodies which bind to a particular epitope on theantigen of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al.(1995) J. Biol. Chem. 270:1388-1394, can be performed to determinewhether the antibody binds an epitope of interest.

Generation of Anti-NRP2 Antibodies from Synthetic Antibody PhageLibraries

In a preferred embodiment, the anti-NRP2 antibodies are selected using aunique phage display approach. The approach involves generation ofsynthetic antibody phage libraries based on single framework template,design of sufficient diversities within variable domains, display ofpolypeptides having the diversified variable domains, selection ofcandidate antibodies with high affinity to target NRP antigen, andisolation of the selected antibodies.

Details of the phage display methods can be found, for example, inWO03/102157 published Dec. 11, 2003.

In one aspect, the antibody libraries can be generated by mutating thesolvent accessible and/or highly diverse positions in at least one CDRof an antibody variable domain. Some or all of the CDRs can be mutatedusing the methods provided herein. In some embodiments, it may bepreferable to generate diverse antibody libraries by mutating positionsin CDRH1, CDRH2 and CDRH3 to form a single library or by mutatingpositions in CDRL3 and CDRH3 to form a single library or by mutatingpositions in CDRL3 and CDRH1, CDRH2 and CDRH3 to form a single library.

A library of antibody variable domains can be generated, for example,having mutations in the solvent accessible and/or highly diversepositions of CDRH1, CDRH2 and CDRH3. Another library can be generatedhaving mutations in CDRL1, CDRL2 and CDRL3. These libraries can also beused in conjunction with each other to generate binders of desiredaffinities. For example, after one or more rounds of selection of heavychain libraries for binding to a target antigen, a light chain librarycan be replaced into the population of heavy chain binders for furtherrounds of selection to increase the affinity of the binders.

Preferably, a library is created by substitution of original amino acidswith variant amino acids in the CDRH3 region of the variable region ofthe heavy chain sequence. The resulting library can contain a pluralityof antibody sequences, wherein the sequence diversity is primarily inthe CDRH3 region of the heavy chain sequence.

In one aspect, the library is created in the context of the humanizedantibody 4D5 sequence, or the sequence of the framework amino acids ofthe humanized antibody 4D5 sequence. Preferably, the library is createdby substitution of at least residues 95-100a of the heavy chain withamino acids encoded by the DVK codon set, wherein the DVK codon set isused to encode a set of variant amino acids for every one of thesepositions. An example of an oligonucleotide set that is useful forcreating these substitutions comprises the sequence (DVK)₇. In someembodiments, a library is created by substitution of residues 95-100awith amino acids encoded by both DVK and NNK codon sets. An example ofan oligonucleotide set that is useful for creating these substitutionscomprises the sequence (DVK)₆ (NNK). In another embodiment, a library iscreated by substitution of at least residues 95-100a with amino acidsencoded by both DVK and NNK codon sets. An example of an oligonucleotideset that is useful for creating these substitutions comprises thesequence (DVK)₅ (NNK). Another example of an oligonucleotide set that isuseful for creating these substitutions comprises the sequence (NNK)₆.Other examples of suitable oligonucleotide sequences can be determinedby one skilled in the art according to the criteria described herein.

In another embodiment, different CDRH3 designs are utilized to isolatehigh affinity binders and to isolate binders for a variety of epitopes.The range of lengths of CDRH3 generated in this library is 11 to 13amino acids, although lengths different from this can also be generated.H3 diversity can be expanded by using NNK, DVK and NVK codon sets, aswell as more limited diversity at N and/or C-terminal.

Diversity can also be generated in CDRH1 and CDRH2. The designs ofCDR-H1 and H2 diversities follow the strategy of targeting to mimicnatural antibodies repertoire as described with modification that focusthe diversity more closely matched to the natural diversity thanprevious design.

For diversity in CDRH3, multiple libraries can be constructed separatelywith different lengths of H3 and then combined to select for binders totarget antigens. The multiple libraries can be pooled and sorted usingsolid support selection and solution sorting methods as describedpreviously and herein below. Multiple sorting satrategies may beemployed. For example, one variation involves sorting on target bound toa solid, followed by sorting for a tag that may be present on the fusionpolypeptide (eg. anti-gD tag) and followed by another sort on targetbound to solid. Alternatively, the libraries can be sorted first ontarget bound to a solid surface, the eluted binders are then sortedusing solution phase binding with decreasing concentrations of targetantigen. Utilizing combinations of different sorting methods providesfor minimization of selection of only highly expressed sequences andprovides for selection of a number of different high affinity clones.

High affinity binders for the target NRP2 antigen can be isolated fromthe libraries. Limiting diversity in the H1/H2 region decreasesdegeneracy about 10⁴ to 10⁵ fold and allowing more H3 diversity providesfor more high affinity binders. Utilizing libraries with different typesof diversity in CDRH3 (eg. utilizing DVK or NVT) provides for isolationof binders that may bind to different epitopes of a target antigen.

Of the binders isolated from the pooled libraries as described above, ithas been discovered that affinity may be further improved by providinglimited diversity in the light chain. Light chain diversity is generatedin this embodiment as follows in CDRL1: amino acid position 28 isencoded by RDT; amino acid position 29 is encoded by RKT; amino acidposition 30 is encoded by RVW; amino acid position 31 is encoded by ANW;amino acid position 32 is encoded by THT; optionally, amino acidposition 33 is encoded by CTG; in CDRL2: amino acid position 50 isencoded by KBG; amino acid position 53 is encoded by AVC; andoptionally, amino acid position 55 is encoded by GMA; in CDRL3: aminoacid position 91 is encoded by TMT or SRT or both; amino acid position92 is encoded by DMC; amino acid position 93 is encoded by RVT; aminoacid position 94 is encoded by NHT; and amino acid position 96 isencoded by TWT or YKG or both.

In another embodiment, a library or libraries with diversity in CDRH1,CDRH2 and CDRH3 regions is generated. In this embodiment, diversity inCDRH3 is generated using a variety of lengths of H3 regions and usingprimarily codon sets XYZ and NNK or NNS. Libraries can be formed usingindividual oligonucleotides and pooled or oligonucleotides can be pooledto form a subset of libraries. The libraries of this embodiment can besorted against target bound to solid. Clones isolated from multiplesorts can be screened for specificity and affinity using ELISA assays.For specificity, the clones can be screened against the desired targetantigens as well as other nontarget antigens. Those binders to thetarget NRP1 antigen can then be screened for affinity in solutionbinding competition ELISA assay or spot competition assay. High affinitybinders can be isolated from the library utilizing XYZ codon setsprepared as described above. These binders can be readily produced asantibodies or antigen binding fragments in high yield in cell culture.

In some embodiments, it may be desirable to generate libraries with agreater diversity in lengths of CDRH3 region. For example, it may bedesirable to generate libraries with CDRH3 regions ranging from about 7to 19 amino acids.

High affinity binders isolated from the libraries of these embodimentsare readily produced in bacterial and eukaryotic cell culture in highyield. The vectors can be designed to readily remove sequences such asgD tags, viral coat protein component sequence, and/or to add inconstant region sequences to provide for production of full lengthantibodies or antigen binding fragments in high yield.

A library with mutations in CDRH3 can be combined with a librarycontaining variant versions of other CDRs, for example CDRL1, CDRL2,CDRL3, CDRH1 and/or CDRH2. Thus, for example, in one embodiment, a CDRH3library is combined with a CDRL3 library created in the context of thehumanized 4D5 antibody sequence with variant amino acids at positions28, 29, 30, 31, and/or 32 using predetermined codon sets. In anotherembodiment, a library with mutations to the CDRH3 can be combined with alibrary comprising variant CDRH1 and/or CDRH2 heavy chain variabledomains. In one embodiment, the CDRH1 library is created with thehumanized antibody 4D5 sequence with variant amino acids at positions28, 30, 31, 32 and 33. A CDRH2 library may be created with the sequenceof humanized antibody 4D5 with variant amino acids at positions 50, 52,53, 54, 56 and 58 using the predetermined codon sets.

Anti-NRP2 Antibody Mutants

The anti-NRP2 antibody generated from phage libraries can be furthermodified to generate antibody mutants with improved physical, chemicaland or biological properties over the parent antibody. Where the assayused is a biological activity assay, the antibody mutant preferably hasa biological activity in the assay of choice which is at least about 10fold better, preferably at least about 20 fold better, more preferablyat least about 50 fold better, and sometimes at least about 100 fold or200 fold better, than the biological activity of the parent antibody inthat assay. For example, an anti-NRP1 antibody mutant preferably has abinding affinity for NRP which is at least about 10 fold stronger,preferably at least about 20 fold stronger, more preferably at leastabout 50 fold stronger, and sometimes at least about 100 fold or 200fold stronger, than the binding affinity of the parent anti-NRPantibody.

To generate the antibody mutant, one or more amino acid alterations(e.g. substitutions) are introduced in one or more of the hypervariableregions of the parent antibody. Alternatively, or in addition, one ormore alterations (e.g. substitutions) of framework region residues maybe introduced in the parent antibody where these result in animprovement in the binding affinity of the antibody mutant for theantigen from the second mammalian species. Examples of framework regionresidues to modify include those which non-covalently bind antigendirectly (Amit et al. (1986) Science 233:747-753); interact with/effectthe conformation of a CDR (Chothia et al. (1987) J. Mol. Biol.196:901-917); and/or participate in the V_(L)-V_(H) interface (EP 239400B1). In certain embodiments, modification of one or more of suchframework region residues results in an enhancement of the bindingaffinity of the antibody for the antigen from the second mammalianspecies. For example, from about one to about five framework residuesmay be altered in this embodiment of the invention. Sometimes, this maybe sufficient to yield an antibody mutant suitable for use inpreclinical trials, even where none of the hypervariable region residueshave been altered. Normally, however, the antibody mutant will compriseadditional hypervariable region alteration(s).

The hypervariable region residues which are altered may be changedrandomly, especially where the starting binding affinity of the parentantibody is such that such randomly produced antibody mutants can bereadily screened.

One useful procedure for generating such antibody mutants is called“alanine scanning mutagenesis” (Cunningham and Wells (1989) Science244:1081-1085). Here, one or more of the hypervariable region residue(s)are replaced by alanine or polyalanine residue(s) to affect theinteraction of the amino acids with the antigen from the secondmammalian species. Those hypervariable region residue(s) demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other mutations at or for the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. The ala-mutants produced this way arescreened for their biological activity as described herein.

Normally one would start with a conservative substitution such as thoseshown below under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity (e.g. bindingaffinity), then more substantial changes, denominated “exemplarysubstitutions” in the following table, or as further described below inreference to amino acid classes, are introduced and the productsscreened.

Preferred Substitutions:

Original Exemplary Preferred Residue Substitutions Substitutions Ala (A)val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arggln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly(G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met;ala; phe; leu norleucine Leu (L) norleucine; ile; val; met; ala; ile pheLys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val;ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; ala; leu norleucine

Even more substantial modifications in the antibodies biologicalproperties are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr, asn, gln;

(3) acidic: asp, glu;

(4) basic: his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

In another embodiment, the sites selected for modification are affinitymatured using phage display (see above).

Nucleic acid molecules encoding amino acid sequence mutants are preparedby a variety of methods known in the art. These methods include, but arenot limited to, oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared mutantor a non-mutant version of the parent antibody. The preferred method formaking mutants is site directed mutagenesis (see, e.g., Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488).

In certain embodiments, the antibody mutant will only have a singlehypervariable region residue substituted. In other embodiments, two ormore of the hypervariable region residues of the parent antibody willhave been substituted, e.g. from about two to about ten hypervariableregion substitutions.

Ordinarily, the antibody mutant with improved biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of either the heavyor light chain variable domain of the parent antibody, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, and most preferably at least 95%. Identity or similarity withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e sameresidue) or similar (i.e. amino acid residue from the same group basedon common side-chain properties, see above) with the parent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

Following production of the antibody mutant, the biological activity ofthat molecule relative to the parent antibody is determined. As notedabove, this may involve determining the binding affinity and/or otherbiological activities of the antibody. In a preferred embodiment of theinvention, a panel of antibody mutants is prepared and screened forbinding affinity for the antigen such as NRP1 or a fragment thereof. Oneor more of the antibody mutants selected from this initial screen areoptionally subjected to one or more further biological activity assaysto confirm that the antibody mutant(s) with enhanced binding affinityare indeed useful, e.g. for preclinical studies.

The antibody mutant(s) so selected may be subjected to furthermodifications, oftentimes depending on the intended use of the antibody.Such modifications may involve further alteration of the amino acidsequence, fusion to heterologous polypeptide(s) and/or covalentmodifications such as those elaborated below. With respect to amino acidsequence alterations, exemplary modifications are elaborated above. Forexample, any cysteine residue not involved in maintaining the properconformation of the antibody mutant also may be substituted, generallywith serine, to improve the oxidative stability of the molecule andprevent aberrant cross linking. Conversely, cysteine bond(s) may beadded to the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment). Another typeof amino acid mutant has an altered glycosylation pattern. This may beachieved by deleting one or more carbohydrate moieties found in theantibody, and/or adding one or more glycosylation sites that are notpresent in the antibody. Glycosylation of antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Addition of glycosylation sites to theantibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Vectors, Host Cells and Recombinant Methods

The anti-Nrp2 antibodies of the invention can be produced recombinantly,using techniques and materials readily obtainable.

For recombinant production of an anti-NRP2 antibody, the nucleic acidencoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated or synthethized usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to DNAs encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders. For yeast secretion thenative signal sequence may be substituted by, e.g., the yeast invertaseleader, a factor leader (including Saccharomyces and Kluyveromycesα-factor leaders), or acid phosphatase leader, the C. albicansglucoamylase leader, or the signal described in WO 90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody, wild-type DHFR protein, and another selectable markersuch as aminoglycoside 3′-phosphotransferase (APH) can be selected bycell growth in medium containing a selection agent for the selectablemarker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al. (1979) Nature 282:39). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones (1977) Genetics 85:12. The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg (1990) Bio/Technology8:135. Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al. (1991) Bio/Technology 9:968-975.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodynucleic acid. Promoters suitable for use with prokaryotic hosts includethe phoA promoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phos-phate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and most preferablySimian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al. (1982) Nature 297:598-601 on expressionof human β-interferon cDNA in mouse cells under the control of athymidine kinase promoter from herpes simplex virus. Alternatively, therous sarcoma virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv (1982) Nature 297:17-18 on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al. (1977) J. Gen Virol. 36:59); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/−DHFR(CHO, Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA77:4216); mouse sertoli cells (TM4, Mather (1980) Biol. Reprod.23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al. (1982) Annals N.Y. Acad.Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (HepG2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980)Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Antibody Purification

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, isremoved, for example, by centrifugation or ultrafiltration. Carter etal. (1992) Bio/Technology 10:163-167 describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al. (1983) J. Immunol. Meth. 62:1-13). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al. (1986)EMBO J. 5:15671575). The matrix to which the affinity ligand is attachedis most often agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Pharmaceutical Formulations

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent, an anticancer agent, an anti-angiogenic agent, an anti-neoplasticagent, a cytotoxic agent and/or a chemotherapeutic agent. Such moleculesare suitably present in combination in amounts that are effective forthe purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Therapeutic Uses

It is contemplated that the antibodies of the present invention may beused to treat a mammal. In one embodiment, the antibody is administeredto a nonhuman mammal for the purposes of obtaining preclinical data, forexample. Exemplary nonhuman mammals to be treated include nonhumanprimates, dogs, cats, rodents and other mammals in which preclinicalstudies are performed. Such mammals may be established animal models fora disease to be treated with the antibody or may be used to studytoxicity of the antibody of interest. In each of these embodiments, doseescalation studies may be performed in the mammal. Where the antibody isan anti-NRP2 antibody, it may be administered to a host rodent in asolid tumor model, for example.

In addition, or in the alternative, the antibody is used to treat ahuman, e.g. a patient suffering from a disease or disorder who couldbenefit from administration of the antibody.

The present invention encompasses the prevention and treatment oftumoral lymphangiogenesis, the prevention and treatment of tumormetastasis and anti-angiogenic cancer therapy, a novel cancer treatmentstrategy aimed at inhibiting the development of tumor blood vesselsrequired for providing nutrients to support tumor growth. The inventionspecifically includes inhibiting the neoplastic growth of tumor at theprimary site as well as preventing and/or treating metastasis of tumorsat the secondary sites, therefore allowing attack of the tumors by othertherapeutics. Examples of cancer to be treated (including prevention)herein include, but are not limited to, carcinoma, lymphoma, blastoma,sarcoma, and leukemia. More particular examples of such cancers includesquamous cell cancer, lung cancer (including small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung), cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer (including gastrointestinal cancer),pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer, as well as B-cell lymphoma (including low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. More particularly,cancers that are amenable to treatment by the antibodies of theinvention include breast cancer, colorectal cancer, rectal cancer,non-small cell lung cancer, non-Hodgkins lymphoma (NHL), renal cellcancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissuesarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer,melanoma, ovarian cancer, mesothelioma, and multiple myeloma.

It is contemplated that when used to treat various diseases such astumors, the antibodies of the invention can be combined with othertherapeutic agents suitable for the same or similar diseases. When usedfor treating cancer, antibodies of the present invention may be used incombination with conventional cancer therapies, such as surgery,radiotherapy, chemotherapy or combinations thereof.

In certain aspects, other therapeutic agents useful for combinationcancer therapy with the antibody of the invention include otheranti-angiogenic agents. Many anti-angiogenic agents have been identifiedand are known in the arts, including those listed by Carmeliet and Jain(2000).

In one aspect, the antibody of the invention is used in combination witha VEGF antagonist or a VEGF receptor antagonist such as anti-VEGFantibodies, VEGF variants, soluble VEGF receptor fragments, aptamerscapable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies,inhibitors of VEGFR tyrosine kinases and any combinations thereof.Alternatively, or in addition, two or more anti-NRP1 antibodies may beco-administered to the patient. In a more preferred embodiment, theanti-NRP1^(A) or anti-NRP^(B) antibody of the invention is used incombination with an anti-VEGF antibody to generate additive orsynergistic effects. Preferred anti-VEGF antibodies include those thatbind to the same epitope as the anti-hVEGF antibody A4.6.1. Morepreferably the anti-VEGF antibody is bevacizumab or ranibizumab.

In some other aspects, other therapeutic agents useful for combinationtumor therapy with the antibody of the invention include antagonist ofother factors that are involved in tumor growth, such as EGFR, ErbB2(also known as Her2) ErbB3, ErbB4, or TNF. Preferably, the anti-NRP1antibody of the invention can be used in combination with small moleculereceptor tyrosine kinase inhibitors (RTKIs) that target one or moretyrosine kinase receptors such as VEGF receptors, FGF receptors, EGFreceptors and PDGF receptors. Many therapeutic small molecule RTKIs areknown in the art, including, but are not limited to, vatalanib (PTK787),erlotinib (TARCEVA®), OSI-7904, ZD6474 (ZACTIMA®), ZD6126 (ANG453),ZD1839, sunitinib (SUTENT®), semaxanib (SU5416), AMG706, AG013736,Imatinib (GLEEVEC®), MLN-518, CEP-701, PKC-412, Lapatinib (GSK572016),VELCADE®, AZD2171, sorafenib (NEXAVAR®), XL880, and CHIR-265.

The anti-Nrp antibody of the invention, either alone or in combinationwith a second therapeutic agent (such as an anti-VEGF antibody) can befurther used in combination with one or more chemotherapeutic agents. Avariety of chemotherapeutic agents may be used in the combined treatmentmethods of the invention. An exemplary and non-limiting list ofchemotherapeutic agents contemplated is provided herein under“Definition”.

When the anti-Nrp antibody is co-administered with a second therapeuticagent, the second therapeutic agent may be administered first, followedby the anti-Nrp antibody. However, simultaneous administration oradministration of the anti-Nrp antibody first is also contemplated.Suitable dosages for the second therapeutic agent are those presentlyused and may be lowered due to the combined action (synergy) of theagent and anti-Nrp antibody.

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of disease to be treated, the severityand course of the disease, whether the antibody is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. The antibody is suitably administered to thepatient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 50mg/kg (e.g. 0.1-20 mg/kg) of antibody is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. In a preferred aspect, theantibody of the invention is administered every two to three weeks, at adose ranged from about 5 mg/kg to about 15 mg/kg. More preferably, suchdosing regimen is used in combination with a chemotherapy regimen as thefirst line therapy for treating metastatic colorectal cancer. In someaspects, the chemotherapy regimen involves the traditional high-doseintermittent administration. In some other aspects, the chemotherapeuticagents are administered using smaller and more frequent doses withoutscheduled breaks (“metronomic chemotherapy”). The progress of thetherapy of the invention is easily monitored by conventional techniquesand assays.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as used hereinbefore or about from 1 to99% of the heretofore employed dosages. Generally, alleviation ortreatment of a disease or disorder involves the lessening of one or moresymptoms or medical problems associated with the disease or disorder. Inthe case of cancer, the therapeutically effective amount of the drug canaccomplish one or a combination of the following: reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., to decrease to someextent and/or stop) cancer cell infiltration into peripheral organs;inhibit tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. In someembodiments, a composition of this invention can be used to prevent theonset or reoccurrence of the disease or disorder in a subject or mammal

The following examples are intended merely to illustrate the practice ofthe present invention and are not provided by way of limitation. Thedisclosures of all patent and scientific literatures cited herein areexpressly incorporated in their entirety by reference.

EXAMPLES Example 1 Generation and Characterization of Anti-Nrp2^(B)Antibodies

Anti-Nrp2^(B) was isolated from human synthetic antibody phage librariesas previously described (Lee et al., J Mol Biol 340, 1073-1093 (2004)).In brief, phage-displayed synthetic antibody libraries were built on asingle human framework by introducing synthetic diversity atsolvent-exposed positions within the heavy chaincomplementarity-determining regions (CDRs). To improve libraryperformance, monovalent and bivalent antigen-binding fragment (Fab)libraries were constructed, and explored different CDR-H3 diversities byvarying the amino acid composition and CDR length. The library was thenexpanded by increasing the variability of CDR-H3 length and usingtailored codons that mimicked the amino acid composition of naturalCDR-H3 sequences. Using these libraries with completely synthetic CDRsdisplayed on a single scaffold high affinity antibodies were generated.For further details of strategies and methods for generating syntheticantibody libraries with single template, see, for example, WO03/102157published Dec. 11, 2003, the entire disclosure of which is expresslyincorporated herein by reference.

The selection procedures for anti-Nrp clones consisted of variouscombinations of solid-supported and solution-binding sortings that areknown in the art. In solid-supported sortings, the antibody phagelibrary was panned with target antigen coated on NUNC 96-well Maxisorpimmunoplate at concentration of 5 ug/ml. In solution-binding sortingmethod, phage library was incubated with decreasing concentration ofbiotinylated antigen in solution, which then was captured by neutravidincoated on the 96-well Maxisorp plate (2-5 μg/ml). Decreasingconcentration allowed more stringency in panning to fish for tighterbinders.

As the result of combining solid-supported and solution-bindingsortings, one clone from a V_(H) Library (YW68.4) and another from aV_(H)V_(L) Library (YW126.20), were identified as NRP-2 binders. Aseries of in vitro assays were conducted to examine properties andactivities of the selected novel anti-NRP antibodies, including bindingaffinity assays (such as BIAcore) and blocking assays (such asSemaphorin induced growth cone collapse assay and HUVEC assays).

The CDRs of naïve clones were engineered to improve its affinity andstability and anti-Nrp2 antibodies YW68.4.2 and YW68.4.2.36 weregenerated. CHO cells expressed mNrp2 (a1a2b1b2)-His, hNrp2 (a1a2b1b2)-Fcfusion protein, and insect cells expressed hNrp2 (b1b2) were used forantibody screening and characterization. The V_(L) and V_(H) regions ofanti-Nrp2^(B) phage antibody (originally designated YW68.4.2.36) werecloned into mammalian expression vector respectively. Anti-Nrp2^(B)human IgG1 or mIgG2a was expressed in mammalian CHO cells and purifiedwith protein A affinity column.

The amino acid sequences of anti-Nrp2^(B) antibodies YW68.4.2, andYW68.4.2.36 are shown in FIG. 10. The amino acid sequence ofanti-Nrp2^(A) antibody YW126.20 is shown in FIG. 11. The alignment ofthe light chain variable domain sequence of anti-Nrp2^(A) antibodyYW126.20 with human κ1 sequence is shown in FIG. 12. The alignment ofanti-Nrp2^(A) antibody YW126.20 heavy chain variable domain sequencewith human III (hum III) sequence is shown in FIG. 13.

In the following experiments, anti-Nrp2^(B) antibody YW68.4.2.36 wasused, which will be hereinafter referred to as <<Anti-Nrp2^(B)>>. Thisantibody was targeted to the coagulation V/VII factor (b1-b2) domains ofNrp2 (FIG. 1A), as these domains are required for VEGFC binding toneuropilins (Karpanen et al., Faseb J20, 1462-1472 (2006)). In addition,this antibody binds with similar Affinity to murine and human Nrp2 butdoes not bind Nrp1 (FIG. 1B). It has been confirmed that anti-Nrp2^(B)bids exclusively to the b1-b2 domains and does nto bind t th CUB (a1-a2)domains of human Nrp2, which are primarily responsible for semaphorinbinding (Chen et al., Neuron 21, 1283-1290 (1998); Giger et al., Neuron21, 1079-1092 (1998)).

To determine binding affinities of anti-Nrp2^(B) IgG1 antibodies,surface plasmon resonance (SRP) measurement with a BIAcore™-3000instrument was used. First of all, anti-Nrp2^(B) human IgGs werecaptured by CM5 biosensor chips coated rabbit anti-human IgG to achieveapproximately 200 response units (RU). For kinetics measurements,two-fold serial dilutions of mouse or human Nrp2 (a1a2b1b2) (0.5 nM to250 nM) were injected separately in PBT buffer (PBS with 0.05% (v/v)Tween 20) at 25° C. with a flow rate of 30 μl/min. Association rates(k_(on)) and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2). The equilibrium dissociation constant (K_(D)) was calculated asthe ratio k_(off)/k_(on).

Anti-Nrp2^(B) binds murine Nrp with a K_(d) of 4.9 nM and to human Nrp2with a K_(d) of 5.3 nM as assessed by surface plasmon resonancemeasurement.

The ability of Anti-Nrp2^(B) to block binding of VEGFC to Nrp2 wastested in both ELISA format and in cell-based binding assays.

For ELISA-based binding specificity tests, three-fold serial dilutionsof Anti-Nrp2^(B) IgGs (0.002 nM to 500 nM) in PBST buffer (PBT bufferwith 0.5% (w/v) BSA) were incubated with 1 μg/ml antigen-coated 96-wellMaxisorp plates for at least 1 hr, and the plates were washed with PBTbuffer. Bound antibodies were detected with anti-human antibody HRPconjugates diluted 1:2500 in PBST buffer, developed with TMB substratefor approximately 5 minutes, quenched with 1M H₃PO₄, and readspectrophotometrically at 450 nm.

To evaluate blocking Nrp2 from binding VEGF, three-fold serial dilutionsof anti-Nrp2^(B) IgGs were first incubated with 96-well Maxisorp platecoated NRP2-Fc (5 μg/ml) in PBST buffer for 2 hr, following by addingbiotinylated VEGF₁₆₅ or VEGFC (full length) for 15 minutes. The amountof biotinylated VEGF binding to Nrp2 was detected by streptavidin-HRPconjugates.

To assess cellular binding, biotinylated VEGF₁₆₅ or VEGFC binding toLECs was carried out as previously described (Jia et al., J Biol Chem281, 13493-13502 (2006)) and binding detected by streptavidin-alkalinephosphatase conjugates. Sema3F binding was performed as previouslydescribed (Chen et al., 1998, supra).

Anti-Nrp2^(B) strongly blocked VEGFC binding to Nrp2 (FIG. 1C) andHEK-293 cells transfected with full length Nrp2. As Nrp2 can also bindVEGF (Gluzman-Poltorak et al., J Biol Chem 275, 29922 (2000)), likelyutilizing the same domains, the ability of anti-Nrp2^(B) to blockVEGF₁₆₅ binding to Nrp2 was also tested. Anti-Nrp2^(B) also stronglyblocks VEGF₁₆₅ binding to Nrp2 (FIG. 1D) with a similar IC₅₀ (0.1 nM).However, Anti-Nrp2^(B) was not able to block binding of Sema3F to LECs(FIG. 1 ^(E)) which strongly express Nrp2 (FIG. 14). These results areconsistent with previous observations that the a1-a2 domains areprimarily responsible for semaphorin binding and the b1-b2 domains forVEGF binding (FIG. 1A).

Example 2 Anti-Nrp2^(B) Blocks Selective VEGFC-Mediated Functions InVitro Materials and Methods

Cell Cultures

HMVEC-dLyAd—Human Dermal Lymphatic Microvascular Endothelial Cells(LECs) and HUVECS were purchased from Cambrex and cultured in EGM-2medium (Cambrex). C6 LacZ cells were purchased from ATCC. 66C14 were akind gift from Dr. Fred R Miller. Tumor cells were cultured in DMEM(Gibco) supplemented with 10% FBS. All cells were maintained at 37° C.in a 5% CO₂, 95% humidity incubator.

Cell Proliferation Assay

A 96-well black-clear bottom plate (VWR) was coated with 5 μg/mlFibronectin (Invitrogen) at 37° C. for 2 hours. LEC's were harvested andresuspended in assay medium (0.1% BSA, EGM-2) 3000 cells/100 ul andadded to wells. Cells were incubated at 37° C. for 16 hours. BrdUlabeling solution (Cell Proliferation ELISA kit; Roche) was added andthe cells were incubated for a further 24 hours at 37° C. BrdUincorporation was determined by chemiluminescence immunoassay. (6 wellsper condition).

Results

The role of Nrp2 in VEGFC mediated migration and proliferation wasexamined. These are key cellular activities induced by VEGFC (Joukov etal., Embo J 16, 3898-3911 (1997)). LECs have been previously shown to behighly responsive to VEGFC (Makinen et al., Embo J 20, 4762-4773 (2001);Veikkola et al., Faseb 17, 2006-2013 (2003); Whitehurst et al., LymphatRes Biol 4, 119-142 (2006)). Using a transwell system, human LECs wereintroduced into the top chamber while VEGFC was added to the bottomchamber to promote migration. LECs that migrated to the bottom were thenfixed, stained (FIG. 2A) and quantified (FIG. 2B). VEGFR3 extracellulardomain protein (ECD), comprising the first three (ligand binding) Igdomains of VEGFR3 was used as a positive control to block VEGFC drivenmigration in this and subsequent experiments (Makinen et al., Nat Med 7,199-205 (2001)). To determine whether Nrp2 function was required for LECmigration, Anti-Nrp2^(B) mAbs were added to cells in the top chamberimmediately prior to the addition of VEGFC. Anti-Nrp2^(B) was able tosignificantly reduce VEGFC mediated LEC migration (FIG. 2A, 2B;p=0.004). The level of inhibition was less than that seen withVEGFR3ECD, which completely inhibited VEGFC mediated LEC migration (FIG.2A, 2B; p<0.001 versus control; p=0.002 versus Anti-Nrp2^(B)). Similarresults were obtained using another VEGFC responsive primary cell line,HUVECs.

As Anti-Nrp2^(B) also blocked VEGF₁₆₅ binding to Nrp2, the role of Nrp2in modulating VEGF₁₆₅ mediated migration was evaluated. VEGF₁₆₅ stronglyinduced migration in LECs as previously described (Hirakawa et al., Am JPathol 162, 575-586 (203); Hong et al., Faseb J 18, 1111-1113 (2004);Makinen et al., Embo J20, 4762-4773 (2001); Veikkola et al., Faseb J 17,2006-2013 (2003)). A cross-species reactive anti-VEGF antibody (B20.4.1)was used as a positive control to block this VEGF driven migration(Liang et al., J Biol Chem 281, 951-961 (2006)). Interestingly,Anti-Nrp2^(B) did not have any effect on VEGF₁₆₅ mediated migration(FIG. 2C), possibly due to the presence of Nrp1 (FIG. 14). Blocking Nrp1function utilizing the Anti-Nrp1 mAb, Anti-Nrp1^(B) (Pan et al., CancerCell 11, 53-67 (2007)) dramatically reduced VEGF mediated migration,confirming this hypothesis (FIG. 15). Addition of both Anti-Nrp1^(B) andAnti-Nrp2^(B) did not result in any further inhibition of migration incomparison to the inhibition seen with Anti-Nrp1^(B) alone (FIG. 15),indicating that Nrp2 does not play a role in VEGF₁₆₅ mediated migration.

Next, the effect of Anti-Nrp2^(B) on VEGFC induced LEC proliferation wasinvestigated. Remarkably, Anti-Nrp2^(B) had no effect on LECproliferation whereas VEGFR3ECD provided a strong block (FIG. 16), inagreement with previous reports where Nrp2 siRNA failed to inhibit VEGFCinduced proliferation in endothelial cells (Favier et al., Blood 108,1243-1250 (2006)). Thus, Nrp2 appears to be important for VEGFC drivenmigration but not proliferation.

Then, the ability of Anti-Nrp2^(B) to modulate semaphorin function wastested. We used the hippocampal neuronal growth cone collapse assay,which previously demonstrated that Nrp2 is required for Sema3F mediatedretraction of the actin-rich structures (Pozas et al., 2001). Additionof Anti-Nrp2^(B) did not have any effect on the semaphorin inducedcollapse while addition of either recombinant Nrp2 A1A2 domain or Nrp2ECD inhibited this collapse completely (FIG. 17). This result isconsistent with our previous observation that Anti-Nrp2^(B) does notbind to the semaphoring binding region and does not interfere withSema3F binding to Nrp2. Thus, Anti-Nrp2^(B) acts to block specificaspects of Nrp2 function, inhibiting VEGFC but not VEGF or Sema3Fmediated cellular responses.

Example 3 Anti-Nrp2^(B) Blocks VEGFC-Mediated Lymphagiogenesis In VivoMaterials and Methods

Mouse Corneal Miro-Pocket Assay

Adult CD-1 mice (Charles-River) were anesthetized and a pocket of 2×3 mmwere created 1 mm from the center of the cornea in the epithelium bymicro-dissection as described previously (Polverini et al., MethodsEnzymol 198, 440-450 (1991)). Agents to be tested for lymphangiogenicactivity were immobilized in an inert hydron pellet (2×2 mm). The pelletwas then implanted into the base of the pocket Animals were treated withcontrol antibody (10 mg/kg), Anti-Nrp2^(B) (10 mg/kg) or VEGFR3ECD 25mg/kg IP twice weekly for 2 weeks. Then animals were sacrificed andcorneas dissected. The lymphatics were visualized by whole-mount IHCwith anti-LYVE-1 antibody (R&D Systems 1:500). The corneas werephotographed and LYVE-1 positive lymphatic vessels arising from thelimbus were quantified.

Mouse Skin Vessel Permeability Assay

The backs and flanks of adult C57BL6J female mice were shaved anddivided into 4 teat areas. They were then injected i.v. with 150 μl 0.5%Evan's blue solution. 1 hr after the Evan's blue injection, 20 μl of PBScontaining BSA or hVEGF (7.5 μg/ml) with or without antibody (0.5 mg/ml)was injected intra dermally, randomly on one of the four zones. 1 hrlater, the animals were sacrificed and the skin was dissected out andimaged. Skin samples for each injection zone were cut out and incubatedin formamide solution at 55° C. for 48 hrs to extract the blue dye. Theabsorbance of the solution was then measured with a spectrometer at 600nm.

Results

Having observed a significant reduction in LEC migration by blockingNrp2 in vitro, it was next examined whether Nrp2 was required formodulating VEGFC function in vivo. We studied two well-characterizedVEGFC mediated in vivo activities—adult lymphangiogenesis and vascularpermeability (Cao et al., Circ Res 94, 664-670 (2004); Joukov et al., JBiol Chem 273, 6599-6602 (1998); Kubo et al., Proc Natl Acad Sci USA 99,8868-8873 (2002); Saaristo et al., Faseb J 16, 1041-1049 (2002)). Tostudy lymphangiogenesis, the murine corneal micropocket assay (Kubo etal., 2002, supra) was utilized. In this assay, a pellet of VEGFC wasintroduced into the avascular cornea of an adult mouse. Over 14 days, inresponse to the VEGFC, a dense plexus of lymphatic vessels grew into thecornea from the limbus (FIG. 2E; 12,000 pixels² with VEGFC treatmentversus 2284 pixels² in control). These vessels were labeled by LYVE-1immunohistochemistry (IHC) and subsequently quantified (FIG. 2D).Systemic administration of VEGFR3ECD almost completely blocked thisVEGFC induced lymphangiogenesis (2671 pixels²; p<0.001). Anti-Nrp2^(B)also effectively blocked the corneal lymphangiogenic response (3281pixels²; p<0.001). This block was similar in degree to the blockobserved using VEGFR3ECD (FIG. 2E, 2D; p=0.67).

In order to evaluate vascular permeability, the miles assay (Brkovic andSirois, J Cell Biochem 100, 727-37 (2007); Eriksson et al., Circulation107, 532-1538 (2003)) was used. This assay uses intradermal injection ofVEGFC to induce vascular permeability and intravascular injection ofEvans blue dye as a tracer to detect and quantify permeability incutaneous vessels (FIG. 16). Remarkably, treatment with Anti-Nrp2^(B)had no effect on VEGFC induced permeability, in contrast to the blockobserved with VEGFR3 ECD treatment (p=0.038). These results demonstratethat, consistent with what we observe in vitro, Nrp2 appears to beimportant for selective VEGFC mediated functions in vivo.

Example 4 Anti-Nrp2^(B) Modulates VEGFC Function by Inhibiting Nrp2/VEGFReceptor Complex Formation Materials and Methods

Cell Migration Assay

Migration assays were performed using a modified Boyden chamber with 8μM pore size Falcon 24-multiwell insert system (BD Biosciences). Theplates were coated with 5 ug/ml Fibronectin (Invitrogen) for 2 hours at37° C. Cells in 100 μl assay medium (0.1% BSA, EGM-2) with/withoutantibodies were added to the upper chamber. Chemoattractant was added tothe lower chamber in 500 μl assay medium, and cells were incubated at37° C. for 16 hours. Cells on the upper membrane were removed with asponge swab and cells on the lower surface were fixed in 70% ethanol andstained with Sytox green (Molecular Probes). Images were taken of theentire lower surface of the well, and number of migrated cells counted.(6 wells per condition).

FACS Analysis

Confluent LECs were incubated with control Anti-Nrp2^(B) antibodies (10μg/ml) for 5 min, 2 hrs or 20 hrs at 37° C. Cells were harvested withenzyme free cell dissociation buffer (Gibco), and incubated withbiotinylated antibody at 1:100 in FACS buffer (PBS, 2% FBS, 2 mM EDTA,0.1% sodium azide) containing 5% normal mouse serum, 2% normal rat serumand 10% 10 μg/ml human IgG. Antibodies were biotinylated using theFluoReporter mini-biotin-xx protein labeling kit (Molecular Probes).Cells were then washed with FACS buffer and stained with streptavidin-PE(BD Biosciences). Data was analyzed with the FacsCalibur system (BDBiosciences).

Cell Adhesion Assay

Subconfluent LECs were pre-incubated in 100 μl Medium 199 with controlor Anti-Nrp2^(B) antibodies (10 μg/ml) for 30 min at 37° C., then platedinto NUNC maxisorp flat bottom 96-well plates (eBioscience) coated with1 μg/ml Fibronectin (Roche) at 10,000 cells per well. Plates werecentrifuged for 1 min at 140 g to synchronize contract of cells withsubstrate, and incubated at 37° C. for 30 min. Plates were then washed 3times with PBS, and frozen at −80° C. Cell density was determined withthe CyQuant kit (Molecular Probes).

VEGF Receptor Signaling Assays

Confluent HUVECs were stimulated for 10 minutes with 200 ng/ml of VEGFCin the presence or absence of control or Anti-Nrp2^(B) antibodies. Thecells were lysed and assayed for many mediators know to play a role ifVEGF receptor signaling. VEGFR2 activation was evaluated using totalVEGFR2 and phospho-VEGFR2 ELISA assays (DuoSet IC ELISA kit, R&D).VEGFR3 activation was evaluated using a kinase receptor activation assay(KIRA) with an VEGFR3—293 cell line as previously described (Sadick etal., J Pharm Biomed Anal 19, 883-891 (1999)). Briefly, stable 293 celllines expressing full length Flag tagged human hVEGFR3 were assayed forreceptor phosphorylation following stimulation. 5×10⁴ cells were starvedovernight (DMEM with 0.1% BSA) and then stimulated with 40 ng/ml VEGFA(Genentech South San Francisco, Calif.) or 200 ng/ml VEGFC (GenentechSouth San Francisco, Calif.) for 8 minutes. Cells were lysed in PBScontaining 1% triton and sodium orthovanadate. ELISA plates were coatedwith capture Flag antibody (Sigma St Louis, Mo.). The plates were coated(PBS+1 ug/ml of antibody) overnight and blocked (PBS+0.5% BSA) for 1 hr.After 3 washes (PBS+0.05% Tween 20), lysates were added for 2 hours,washed three times, followed by addition of phospho-detection antibody4G10 (Upstate Lake Placed, N.Y.) for 2 hours. Detection was performedwith HRP antibody (Amersham Piscataway, N.J.) and TMB substrate. Plateswere read at 450 nm. Total AKT, phospho-AKT, total Erk1/2,phospho-Erk1/2, total Src, phospho-Src, total p38 MAPK and phospho-p38MAPK were evaluated using sandwich ELISA kits from BioSource.

Results

The finding that Anti-Nrp2^(B) interferes with VEGFC actions wasconsistent with the fact that it blocks VEGFC binding to Nrp2. Inaddition, the fact that it blocks only selective functions both in vitroand in vivo was highly unexpected. One possible explanation of why adisruption of LEC migration and lymphangiogenesis but not LECproliferation or vascular permeability was observed is thatAnti-Nrp2^(B) may generally inhibit migration, possibly by disruptingLEC adhesion to the extracellular matrix. To test this, the effect ofAnti-Nrp2^(B) on migration induced by a number of LEC motogens wasevaluated. Anti-Nrp2^(B) did not have any effect on migration induced byVEGF (FIG. 2C), HGF (FIG. 3B) or FGF. Therefore Anti-Nrp2^(B) did notgenerally disrupt LEC migration. Furthermore, Anti-Nrp2^(B) did not haveany effect on LEC adhesion to fibronectin or collagen, two extracellularmatrix substrates.

A second possibility is that the Anti-Nrp2^(B) mAb may causeinternalization of Nrp2 (Jaramillo et al., Exp Cell Res 312, 2778-2790(2006)). As Nrp2 forms a complex with VEGFR3, even in the absence ofligand (Favier et al., 2006, supra; Karkkainen and Alitalo, Semin CellDev Biol 13, 9-18 (2002)), this could result in a selectiveco-internalization of VEGFR3, affecting specific VEGFC mediatedfunctions. This possibility was further validated by the finding thatVEGFC driven vascular permeability is mediated by VEGFR2 and not VEGFR3(Joukov et al., 1998, supra). To address this possibility, LECs wereincubated with Anti-Nrp2^(B) at 37° C. for 5 minutes, 2 hours or 20hours and then performed FACS analysis with antibodies against Nrp2,VEGFR2 and VEGFR3 to determine the level of the receptors on the cellsurface. No difference was observed between treatments, suggesting thatAnti-Nrp2^(B) did not cause significant internalization of Nrp2, VEGFR2or VEGFR3 (FIG. 3A).

As Nrp2 has been proposed to augment VEGF receptor signaling (Favier etal., 2006, supra), next, the effect of Anti-Nrp2^(B) on VEGFR2 andVEGFR3 activation was studied, where VEGFC stimulation leads to receptordimerization and auto-phosphorylation. In agreement with the prior invitro and in vivo data, VEGFR3ECD completely blocked VEGFC mediatedVEGFR2 (FIG. 3C; p<0.001) and VEGFR3 phosphorylation. Anti-Nrp2^(B)treatment resulted in a reduction of VEGFR2 (FIG. 3C; p<0.001) andVEGFR3 activation, but to a significantly lesser degree than VEGFR3ECDtreatment (p<0.001). This observation raised the possibility that theselective inhibitory activity of Anti-Nrp2^(B) could be a result ofdifferential requirements of VEGF receptor activation for migration andproliferation, that migration requires higher levels of receptoractivation than proliferation. To address this possibility, the doseresponse of VEGFR2 phosphorylation to VEGFC stimulation was evaluated(FIG. 3C). It was consistently observed that the reduction of VEGFR2phosphorylation caused by Anti-Nrp2^(B) treatment was equivalent to theVEGFR2 phosphorylation obtained by stimulating with 175 ng/ml or 150ng/ml of VEGFC. Then, a dose response analysis of migration to VEGFCstimulation was performed (FIG. 3D). It has been noted that LECsstimulated with 175 ng/ml or 150 ng/ml of VEGFC migrated equivalently tocells stimulated with 200 ng/ml of VEGFC. Indeed, a significantreduction in migration was not observed till VEGFC levels were reducedto 50 ng/ml. We therefore reasoned that the reduction in VEGFR2phosphorylation induced by Anti-Nrp2^(B) was, by itself, insufficient toreduce migration.

We additionally evaluated the effect of Anti-Nrp2^(B) on downstreamsignaling events mediated by VEGF receptors. Treatment withAnti-Nrp2^(B) or stimulation with 175 ng/ml or 150 ng/ml of VEGFC didnot significantly reduce Erk1/2, Akt or p38 MAPK activation whichmodulate VEGFR2 mediated proliferation, permeability and motilityrespectively, similar to what is observed with Nrp1 (Pan et al., 2007).This indicated that Nrp2 might regulate LEC migration andlymphangiogenesis by a mechanism other than enhancing VEGF receptoractivation or downstream signaling.

Lastly, we the effect of Anti-Nrp2^(B) on Nrp2/VEGF receptor complexformation was tested. As reported previously, Nrp2 can becoimmunoprecipitated with VEGFR2 and VEGFR3 in the presence or absenceof VEGFC (Favier et al., 2006 supra; Karpanen et al., 2006, supra) (FIG.3E). This interaction was dramatically reduced by Anti-Nrp2^(B) (FIG.3E). This result suggests that the Nrp2/VEGF receptor complex isimportant for specific VEGFC mediated functions. Furthermore, the roleof Nrp2 is not exclusively to enhance VEGF receptor signaling inresponse to ligand stimulation.

Example 5 Nrp2 is Expressed in Tumor-Associated Lymphatics Materials andMethods

Immunohistochemistry

18 μm tissue sections were cut and mounted onto glass slides. Thesections were incubated 0/N with primary antibody (anti-NRP2^(B) (1:500control staining performed in E12.5 mouse spinal cord where expressionhas been well characterized), anti-LYVE-1 (anti-R&D, 1:200),anti-PECAM-1 (Benton Dickinson, 1:500), anti-PROX-1 (Chemicon, 1:1000),or Ki67 (Neovision 1:100) at 4° C. Samples were then stained with Alexa488 or Alexa 568 secondary antibodies (1:200; Molecular Probes) for 4hrs at RT. Staining with secondary only was used as a control. TUNNELstaining was performed with a commercial kit (Roche). Images werecaptured with a Zeiss Axiophot fluorescence microscope. Blood andlymphatic vessel area was determined from 6 representative images fromeach of 6 tumors per group, evaluated for mean pixel number by ImageJ.

Results

In order to determine if Nrp2 pays a role in adult lymphatic biology,the expression of Nrp2 in adult lymphatics was evaluated. Within thevascular system, Nrp2 expression has been previously described in veinsand lymphatics (Herzog et al., Mech Dev 109, 115-119 (2001); Moyon etal., Development 128, 3359-3370 (2001); Yuan et al., Development 129,4797-4806 (2002)). As described above, LEC in culture strongly expressNrp2 (FIG. 14). However, we were unable to detect Nrp2 by IHC incolonic, LYVE-1 positive lymphatic vessels of normal adult mice (FIG.4A; for positive control staining see FIG. 17). The colon was evaluated,as it has a rich plexus of lymphatic vessels with a fairly stereotypedpattern. We were also unable to detect Nrp2 expression by IHC withinlymphatic vessels of lymph nodes (FIG. 4B) and skin from normal adultmice. These results were confirmed by Nrp2 in situ hybridization (ISH).In contrast, strong Nrp2 expression was observed in LYVE-1+lymphaticvessels in lymph nodes adjacent to orthotopically or heterotopicallytransplanted tumors (FIG. 4C). This was observed with a number of tumorlines including the orthotopically transplanted murine breastadenocarcinoma line, 66c14 (Aslakson and Miller, Cancer Res 52,1399-1405 (1992)) and the heterotopically transplanted rat glioblastomaline, C6 (data not sown). Nrp2 was also observed in peri-tumoral andintra-tumoral lymphatics (FIG. 4D) for a number of tumor lines including66c14, C6 and PC3 (human prostate carcinoma line). This expression wasconfirmed by ISH in a subset of tumor types.

Example 6 Anti-Nrp2^(B) Reduces Lung Metastasis in Multiple Tumor ModelsMaterials and Methods

All animal studies were in accordance with the Guide for the Care andUse of Laboratory Animals, published by the NIH (NIH Publication 85-23,revised 1985). An Institutional Animal Care and Use Committee (IACUC)approved all animal protocols.

Tumor Models

For 66C14, cells were harvested by trypsinization, washed, andresuspended in PBS at a concentration of 2×10⁵ cells in 10 μl PBS. Micewere anesthetized using 75 mg/kg ketamine and 7.5 mg/kg xylazine, and anincision made underneath the right forelimb. 2×10⁵ cells in 10 μl PBSwas injected directly into the exposed 4^(th) mammary fat pad of 6-8week old female balb-C mice. For C6, 2×10⁶ tumor cells in 100 μl PBSwere injected subcutaneously into the right flank of 6-8 week old femalebalb-C nude mice. For both sets of studies, tumor growth was monitored 3times weekly. When tumors reach an average size of 80-120 mm³, mice weresorted to give nearly identical group mean tumor sizes, and treatmentwas started. This was considered day 1 of each study. Animals weretreated with control antibody (10 mg/kg), Anti-Nrp2^(B) (10 mg/kg) orVEGFR3ECD 25 mg/kg IP twice weekly till study termination. All studieswere repeated 3 times to ensure reproduceability.

At study end animals were anethetiszed and perfused with 4% PFA. Tumorswere harvested, cryoprotected and frozen in OCT (Tissue-Tek). Lungs wereinflated via a right ventricular perfusion of 10 ml of PBS followed by4% PFA, and visual counts of metastatic lesions were performed prior toMicro-CT analysis.

SLN metastasis was evaluated by heterotopic implantation of C6 tumorcells into the ear. Briefly, a cohort of animals were pre-treated withcontrol antibody (10 mg/kg) or Anti-Nrp2^(B) (10 mg/kg), one day priorto tumor cell implantation, and twice weekly thereafter. 1×10⁵ C6 lacZCells were injected subdermally into the ear of 70 female balb/c nudemice. Mice were sacrificed at day 3, 6, 9, 13 and 15, and sentinel lymphnodes identified by cutaneous lymphangiography and subsequentlydissected out. Lymph nodes were homogenized and lysate assayed forβ-galactosidase activity (Pierce).

Intradermal lymphangiography was performed on control and tumor bearingmice as follows. 2 μl of evans blue dye (3% by weight) containing 1% 20nm polystyrene fluorescent microbeads (Molecular Probes) was injectedintradermally at the apex of the tumor. Animals were allowed to recoverfor 2 hours and then sacrificed. Tumors were dissected out with care tonot include peritumoral tissue. They were either fixed and thenhistochemically analyzed or incubated in formamide to extract evans blueand quantified with a OD600 measurement by spectrophotometer.

Micro-CT Analysis of Lungs

Lungs were immersed in 10% NBF for 24 hours, then immersed in a 20%solution of an iodine-based x-ray computed tomography contrast agent,Isovue370 (Bracco Diagnostics Inc, Princeton, N.J.), was diluted withPBS for 24 hours. Lungs were then immersed in and perfused via thetrachea cannula with 20 mls of soy bean oil (Sigma-Aldrich, St. Louis,Mo.) at a rate of 0.25 ml/min. The soy bean oil was used to removeexcess contrast agent and provide a background media for imaging.

The mouse lungs were imaged ex-vivo with a VivaCT (SCANCO Medical,Basserdorf, Switzerland) x-ray micro-computed tomography (micro-CT)system. A sagittal scout image, comparable with a conventional planarx-ray, was obtained to define the start and end point for the axialacquisition of a series of micro-CT image slices. The location andnumber of axial images were chosen to provide complete coverage of thelung. The lungs were immersed in soybean oil as the background media.The micro-CT images were generated by operating the x-ray tube at anenergy level of 45 kV, a current of 160 μA and an integration time of450 milliseconds. Axial images were obtained at an isotropic resolutionof 21 μm. The lung tumor estimates (number and volume) were obtained byan semi-automated image analysis algorithm that includes an inspectionstep by a trained reader. Lung tumors appear as a hyper-intense solidmass relative to porous, mesh-like structures of the normal lung. Thisis due to the absorption of the iodine contrast agent by solidstructures (bronchial and aveloi walls, tumors, trachea, medial steinum)contained within the lung, Excess contrast agent was cleared from thefilled air spaces by the oil perfusion step. Potential tumor masses wereextracted by a series of image processing steps. The image analysissoftware was developed in-house. It was written in C++ and employed theAnalyze (AnalyzeDirect Inc., Lenexa, Kans., USA) image analysis softwarefunction libraries. The algorithm employs intensity thresholding,morphological filtering and region-growing to extract all potentialtumors masses. An intensity threshold (1480 Hounsfield Units) wasdetermined by histogram analysis of 5 arbitrary lungs employed foralgorithm development and the optimal threshold was chosen to includetumor voxels and exclude any background signal. Morphological (erosion,dilation) and region-growing operations were applied to connecthyper-intense regions of voxels and to remove any voxels of similardensity found in the thin walls of the bronchioles and aveoli. Theregion growing step requires a minimum volume of 2300 connected voxels(greater than 0.0231 mm³) to be accepted as an object (mass). Theidentified objects were then evaluated by a trained reader with theAnalyze 3D visualization software. Individual objects were accepted orrejected as possible tumors based on the appearance of the object andits location within the lung. Objects were rejected if they resideoutside the lung (ex. mediasteinum, extraneous tissue debris) orresemble a blood-filled vessel. The tumor count, individual tumor volumeand total tumor volume were determined for each lung. This analytictechnique was validated with a well-established tumor metastasis model.Eleven animals with orthotopic transplantation of 4T 1 breast mammaryadenocarcinoma tumor cells were evaluated for lung metastasis by thismicro-CT technique followed by serial histologic analysis of the lungs.Lung tumor volume estimates were highly correlated (r=0.9, p=0.0002)with histological estimates of tumor size (pixel count; FIG. 18).

Results

One major approach to inhibiting metastasis has been via inhibition ofthe VEGFC axis (Chen et al., Cancer Res 65, 9004-9011 (2005); He et al.,J Natl Cancer Inst 94, 819-825 (2002); Krishnan et al., Cancer Res 63,713-722 (2003); Lin et al., Cancer Res 65, 6901-6909 (2005)). Todetermine if blocking Nrp2 function could also modulate the developmentof metastasis, the effects of Anti-Nrp2^(B) treatment on the formationof lung metastasis were teste in two different tumor models—the 66c14,breast cancer model, and the C6 glioblastoma model. 66c14 is a murinemammary adenocarcinoma line derived from a spontaneous mammary tumor ina Balb/c mouse (Aslakson and Miller, Cancer Res 52, 1399-1405 (1992)).These cells express VEGFC and metastasize via the lymphatic system tothe lungs (Aslakson and Miller, 1992, supra). Orthotopic transplantationof these cells in Balb/c mice resulted in reproducible development oftumors and lung metastasis. Anti-Nrp2^(B) treatment did not affect theprimary growth rate of the tumors (FIG. 5A). As VEGFR3ECD diddramatically reduce primary tumor growth rates it was excluded from anyfurther analysis of metastasis. A cohort of animals (N=6 from eachgroup) with similar sized tumors from both treatment arms weresacrificed concurrently, and the lungs were dissected out and inflatedto facilitate analysis for metastatic nodules. Anti-Nrp2^(B) caused adramatic reduction in the average number of visually detected metastaticnodules per lung when compared to control IgG treated animals (FIG. 5B,C), from an average of 3.5 to 0.8 (P=0.03). In order to confirm thisresult and extend our evaluation to metastasis within the lungparenchyma that were not amenable to visual examination, we performed amicro-CT analysis (Li et al., Technol Cancer Res Treat 5, 147-155(2006)) of the lungs after necropsy. This analysis confirmed thatAnti-Nrp2^(B) treated animals had a reduction in the number of lungmetastasis when compared to control treated animals (FIG. 5D-F).However, micro-CT analysis was more sensitive than visual analysisresulting in a larger absolute number of metastatic nodules in bothgroups. Micro-CT also allowed us to determine the total metastaticburden within the lung. Anti-Nrp2^(B) treatment also resulted in areduction of total metastatic volume (0.74 cm³) in comparison to controltreatment (1.78 cm³). Additionally, this analysis verified that the vastmajority of lesions were on the surface of lungs in both control andAnti-Nrp2^(B) treated animals. Therefore, Anti-Nrp2^(B) did not cause areduction in metastasis by shifting the nodules from the surface to thelung parenchyma.

FACS analysis, performed as described in Example 4, indicated that Nrp2,but not VEGFR2 or VEGFR3 was expressed on 66c14 tumor cells. This raisedthe possibility that treatment with Anti-Nrp2^(B) was affecting tumorcell behavior directly to impact metastasis. This was unlikely given thelack of effect on primary tumor growth with Anti-Nrp2^(B) treatment.Additionally, Anti-Nrp2^(B) did not have any effect on tumor cellproliferation, apoptosis or migration in vitro. However, to address thepossibility that the reduction in metastasis was due to effects ofAnti-Nrp2^(B) on tumor cells, we also evaluated the effect ofAnti-Nrp2^(B) on the C6 rat glioblastoma model. These cells do notexpress Nrp2 on their surface to an appreciable degree (FIG. 6E),express VEGFC and are thought to metastasize to the lung via thelymphatic system (Bernstein and Woodard, 1995). Additionally, they havebeen engineered to express b□galactosidase, which can be used as amarker to facilitate detection of tumor cells.

Subcutaneous transplantation of these cells in nude mice resulted inconsistent development of tumors and lung metastasis. Anti-Nrp2^(B)treatment did not affect the primary growth rate of these tumors (FIG.6A). Additionally, VEGFR3ECD did not dramatically reduce primary tumorgrowth rate in this tumor model. This allowed for comparisons of theanti-metastatic effects of VEGFR3ECD and Anti-Nrp2. Again, a cohort ofanimals (N=10) with similar sized tumors from all treatment arms weresacrificed and the lungs were dissected out and inflated to facilitateanalysis for metastatic nodules. Treatment with both, Anti-Nrp2^(B) andVEGFR3ECD, caused a reduction in the average number of visually detectedmetastatic nodules per lung (FIG. 6B, C). The reduction noted withAnti-Nrp2^(B) was comparable to that seen with VEGFR3ECD. Micro-CTanalysis of the lungs confirmed these findings (FIG. 6D) and verifiedthat the vast majority of metastasis were localized to the surface ofthe lungs in all treatment arms. Nodules were confirmed to be metastaticlesions by histology in both tumor models (FIGS. 5H and 6G).Additionally, general necropsy did not reveal nodules on the surface ofother organs in either tumor model.

Although in the foregoing description the invention is illustrated withreference to certain embodiments, it is not so limited. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

All references cited throughout the specification, and the referencescited therein, are hereby expressly incorporated by reference in theirentirety.

What is claimed:
 1. A method for inhibiting lymphatic endothelial cellmigration, comprising administering to a mammalian subject in need aneffective amount of an anti-neuropilin2B (anti-Nrp2B) antibody, or anantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment: i) comprises complementarity determiningregion light chain (CDRL)1, CDRL2, and CDRL3 sequences andcomplementarity determining region heavy chain (CDRH)1, CDRH2, CDRH3sequences of an antibody selected from the group consisting of YW68.4.2and YW68.4.2.36; ii) is a variant of the antibody of (i) having one tofive alterations in the framework regions, wherein the light chainframework regions comprise residues 1-23, 35-49, 57-88 and 98-107 or1-25, 33-49, 53-90 and 97-107 and the heavy chain framework regionscomprise residues 1-30, 36-49, 66-94 and 103-113 or 1-25, 33-52, 56-95and 102-113; or iii) is an affinity matured variant of the antibody of(i).
 2. The method of claim 1 wherein said mammalian subject is a humanpatient.
 3. The method of claim 2 wherein said human patient has beendiagnosed with cancer.
 4. The method of claim 3 wherein said humanpatient has developed or is at risk of developing tumor metastasis. 5.The method of claim 4 wherein said metastasis is in the lymphaticsystem.
 6. The method of claim 4 wherein said metastasis is in a distantorgan.
 7. The method of claim 3 wherein said cancer is selected from thegroup consisting of carcinoma, lymphoma, blastoma, sarcoma, andleukemia.
 8. The method of claim 3 wherein said cancer is selected fromthe group consisting of squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastric cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, liver cancer, prostate cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer,B-cell lymphoma, chronic lymphocytic leukemia (CLL); acute lymphoblasticleukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia;post-transplant lymphoproliferative disorder (PTLD), abnormal vascularproliferation associated with phakomatoses, edema associated with braintumors, and Meigs' syndrome.
 9. The method of claim 8 wherein saidB-cell lymphoma is selected from the group consisting of lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia.
 10. Themethod of claim 1, wherein said variant is an affinity matured variant.11. The method of claim 1, wherein the anti-Nrp2B antibody, or anantigen-binding fragment thereof: i) comprises the heavy and light chainvariable region sequence of an antibody selected from the groupconsisting of YW68.4.2 and YW68.4.2.36; ii) is a variant of the antibodyof (i) having one to five alterations in the framework regions, whereinthe light chain framework regions comprise residues 1-23, 35-49, 57-88and 98-107 or 1-25, 33-49, 53-90 and 97-107 and the heavy chainframework regions comprise residues 1-30, 36-49, 66-94 and 103-113 or1-25, 33-52, 56-95 and 102-113; or iii) is an affinity matured variantof the antibody of (i).
 12. The method of claim 11, wherein theanti-Nrp2B antibody or antigen-binding fragment comprises the sequenceof YW68.4.2 (SEQ ID NO: 1) or the sequence of YW68.4.2.36 (SEQ ID NO:2).
 13. The method of claim 12, wherein the anti-Nrp2B antibodycomprises the sequence of YW68.4.2.36 (SEQ ID NO: 1), or anantigen-binding fragment thereof.
 14. The method of claim 11, whereinthe anti-Nrp2B antibody is selected from the group consisting ofYW68.4.2 and YW68.4.2.36 and antigen-binding fragments thereof.
 15. Themethod of claim 14, wherein the anti-Nrp2B antibody is YW68.4.2.36 (SEQID NO: 1), or an antigen-binding fragment thereof.
 16. The method ofclaim 1, wherein said antibody, antigen-binding fragment or variant, ischimeric or humanized.
 17. The method of claim 1, wherein saidantigen-binding fragment is selected from the group consisting of Fab,F(ab′)₂, and scFv fragments.
 18. The method of claim 1, wherein theanti-Nrp2B antibody, or antigen-binding fragment or variant isbispecific.
 19. The method of claim 18, wherein the anti-Nrp2B antibody,or antigen-binding fragment or variant additionally binds to VEGF.
 20. Amethod for inhibiting tumoral lymphangiogenesis, comprisingadministering to a tumor-bearing mammalian subject an effective amountof an anti-neuropilin2B (anti-Nrp2B) antibody, or an antigen-bindingfragment thereof, wherein the antibody or antigen-binding fragment: i)comprises complementarity determining region light chain (CDRL)1, CDRL2,and CDRL3 sequences and complementarity determining region heavy chain(CDRH)1, CDRH2, CDRH3 sequences of an antibody selected from the groupconsisting of YW68.4.2 and YW68.4.2.36; ii) is a variant of the antibodyof (i) having one to five alterations in the framework regions, whereinthe light chain framework regions comprise residues 1-23, 35-49, 57-88and 98-107 or 1-25, 33-49, 53-90 and 97-107 and the heavy chainframework regions comprise residues 1-30, 36-49, 66-94 and 103-113 or1-25, 33-52, 56-95 and 102-113; or iii) is an affinity matured variantof the antibody of (i).
 21. A method for treating tumor metastasis,comprising administering to a tumor-bearing mammalian subject aneffective amount of an anti-neuropilin2B (anti-Nrp2B) antibody, or anantigen-binding fragment thereof, wherein the antibody orantigen-binding fragment: i) comprises complementarity determiningregion light chain (CDRL)1, CDRL2, and CDRL3 sequences andcomplementarity determining region heavy chain (CDRH)1, CDRH2, CDRH3sequences of an antibody selected from the group consisting of YW68.4.2and YW68.4.2.36; ii) is a variant of the antibody of (i) having one tofive alterations in the framework regions, wherein the light chainframework regions comprise residues 1-23, 35-49, 57-88 and 98-107 or1-25, 33-49, 53-90 and 97-107 and the heavy chain framework regionscomprise residues 1-30, 36-49, 66-94 and 103-113 or 1-25, 33-52, 56-95and 102-113; or iii) is an affinity matured variant of the antibody of(i).
 22. The method of claim 20 or claim 21 wherein said mammaliansubject is a human cancer patient.
 23. The method of claim 22 whereinsaid human cancer patient has developed or is at risk of developingtumor metastasis.
 24. The methor of claim 23 wherein said tumormetastasis is in the lymphatic system.
 25. The method of claim 23wherein said tumor metastasis is in a distant organ.
 26. The method ofclaim 22 wherein said cancer is selected from the group consisting ofcarcinoma, lymphoma, blastoma, sarcoma, and leukemia.
 27. The method ofclaim 22 wherein said cancer is selected from the group consisting ofsquamous cell cancer, small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung, squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric cancer,gastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, B-cell lymphoma,chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);Hairy cell leukemia; chronic myeloblastic leukemia; post-transplantlymphoproliferative disorder (PTLD), abnormal vascular proliferationassociated with phakomatoses, edema associated with brain tumors, andMeigs' syndrome.
 28. The method of claim 27 wherein said B-cell lymphomais selected from the group consisting of low grade/follicularnon-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia.
 29. Themethod of claim 23, wherein said variant is an affinity matured variant.30. The method of claim 23, wherein the anti-Nrp2B antibody, or anantigen-binding fragment thereof: i) comprises the heavy and light chainvariable region sequence of an antibody selected from the groupconsisting of YW68.4.2 and YW68.4.2.36; ii) is a variant of the antibodyof (i) having one to five alterations in the framework regions, whereinthe light chain framework regions comprise residues 1-23, 35-49, 57-88and 98-107 or 1-25, 33-49, 53-90 and 97-107 and the heavy chainframework regions comprise residues 1-30, 36-49, 66-94 and 103-113 or1-25, 33-52, 56-95 and 102-113; or iii) is an affinity matured variantof the antibody of (i).
 31. The method of claim 30, wherein theanti-Nrp2B antibody or antigen-binding fragment comprises the sequenceof YW68.4.2 (SEQ ID NO: 1) or the sequence of YW68.4.2.36 (SEQ ID NO:2).
 32. The method of claim 31, wherein the anti-Nrp2B antibodycomprises the sequence of YW68.4.2.36 (SEQ ID NO: 1), or anantigen-binding fragment thereof.
 33. The method of claim 30, whereinthe anti-Nrp2B antibody is selected from the group consisting ofYW68.4.2 and YW68.4.2.36 and antigen-binding fragments thereof.
 34. Themethod of claim 33, wherein the anti-Nrp2B antibody is YW68.4.2.36 (SEQID NO: 1), or an antigen-binding fragment thereof.
 35. The method ofclaim 23, wherein said antibody, antigen-binding fragment or variant, ischimeric or humanized.
 36. The method of claim 23, wherein saidantigen-binding fragment is selected from the group consisting of Fab,F(ab′)₂, and scFv fragments.
 37. The method of claim 23, wherein theanti-Nrp2B antibody, or antigen-binding fragment or variant isbispecific.
 38. The method of claim 37, wherein the anti-Nrp2B antibody,or antigen-binding fragment or variant additionally binds to VEGF.